Considerable interest in the physiology of proliferation has been generated by human cancers. Cancers often share features with other proliferative cells that are exemplified by the Warburg effect, which involves an altered metabolism with less oxygen uptake and greater lactate secretion. While some of the Warburg effect can be rationalized as production of biosynthetic precursors to fuel proliferation, lactate secretion does not fit this paradigm as it wastes these valuable precursors. In this context, examining proliferation in organisms that are capable of anaerobic pathways other than lactate production can be illuminating. Many animal species, and indeed eukaryotes in general, can carry out other bioenergetic reactions that do not use oxygen. These reactions frequently involve mitochondria and employ such seemingly baroque processes as running the Krebs cycle, the metabolic heart of the cell, backwards. Detailed study of these processes may require development of new animal model systems. For example, colonial marine hydroids frequently exhibit a stage in the life cycle that only undergoes cellular proliferation and never produces gametes. In contrast, traditional animal models such as worms, flies, and mice undergo only limited proliferation before gamete production. The crucial unresolved issues related to the Warburg effect could thus be understood.Interest in the physiology of proliferation has been generated by human proliferative diseases, i.e., cancers. A vast literature exists on the Warburg effect, which is characterized by aerobic glycolysis, diminished oxygen uptake, and lactate secretion. While these features could be rationalized via the production of biosynthetic precursors, lactate secretion does not fit this paradigm, as it wastes precursors. Forming lactate from pyruvate allows for reoxidizing cytosolic NADH, which is crucial for continued glycolysis and may allow for maintaining large pools of metabolic intermediates. Alternatively, lactate production may not be adaptive, but rather reflect metabolic constraints. A broader sampling of the physiology of proliferation, particularly in organisms that could reoxidize NADH using other pathways, may be necessary to understand the Warburg effect. The best-studied metazoans  may not be suitable, as they undergo limited proliferation before initiating meiosis. In contrast, some metazoans  exhibit a stage in the life cycle (the polyp stage) that only undergoes mitotic proliferation and never carries out meiosis (the medusa stage performs this). Such organisms are prime candidates for general studies of proliferation in multicellular organisms and could at least complement the short-generation models of modern biology. Considerable interest in the physiology of proliferation has been generated by human proliferative diseases, i.e., cancers. A vast literature exists on the so-called Warburg effect ,6,7,8,9,+) to continue. Apart from some vertebrates, very few metazoans primarily use this pathway to maintain redox balance in the absence of molecular oxygen [Pyruvate may be reduced to lactate to reoxidize cytosolic NADH and allow for glycolysis  depend on large amounts of ATP. During proliferation, why is pyruvate not processed in the Krebs cycle and electron transport chain to maximize ATP formation?Further, the best-studied metazoans  undergo limited proliferation before initiating the sexual phase of the life cycle. In contrast, some metazoans  exhibit a stage in the life cycle (the polyp stage) that only undergoes mitotic proliferation and never carries out meiosis to form gametes (this is conducted in the medusa stage). The proliferative polyp stage can be extraordinarily long-lived . Such organisms are prime candidates for general studies of proliferation in multicellular organisms and could minimally complement the short-generation models of modern biology.Two robust fields of intellectual inquiry have existed independently for some time. On the one hand, the Warburg effect is studied by biomedical scientists using human cells. On the other hand, organismal biologists and physiologists investigate the anaerobic metabolism using a host of nonmodel organisms. We attempt to unite these distinct fields of inquiry here. It is not our goal to extend the boundaries of either field or to elaborate their rich details. Both fields have developed large literatures and have great numbers of active researchers. Our goal is to connect these fields for useful synergisms.Further, to broaden the study of proliferation, we outline the biological features that should be included in these more general studies. First, it is essential to examine how the Warburg effect manifests in organisms that are capable of a sophisticated (for eukaryotes) anaerobic metabolism. What are the metabolic features of proliferative cells and organisms that are capable of reoxidizing NADH using pathways other than lactate production or the canonical electron transport chain? Second, the model systems of modern biology were mostly developed to study genetics. These model organisms rapidly complete the proliferative phase of the life cycle and may be largely postmitotic as adults. We suggest including organisms with life cycle stages that are entirely mitotic and long-lived. Cancer cells proliferate mitotically, but they never undergo meiosis. Hence, it is not unreasonable to develop models of proliferation in general and cancer in particular that have the same characteristics. Lastly, keeping in mind the admonishment from Martin et al.  (p. 5): In Metazoa, how generally do proliferative cells and organisms exhibit the Warburg effect?Do high rates of proliferation result in lactate formation or are alternative pathways used to reoxidize NADH?Why is NADH not reoxidized using the entire electron transport chain with oxygen as a terminal electron acceptor?This research program provides answers to various questions related to the Warburg effect that have remained unresolved for nearly a century, including the following:In terms of structure, function, life cycle, genes, and genomes, eukaryotes are remarkably complex. On the other hand, in contrast to prokaryotes, both aerobic and anaerobic metabolism is highly stereotyped in eukaryotes ,11,12. AUnder more long-lasting anaerobic conditions, many metazoans ,11,12 caMartin et al.  provide In anaerobic mitochondria, malate dismutation uses rhodoquinone (RQ) due to its strong electron donor potential relative to ubiquinone (UQ). The alternative splicing of a specific exon provides the capability to synthesize RQ . Two cri+. Lunt and Vander Heiden [The modern conceptualization of the Warburg effect focuses on aerobic glycolysis and lactate secretion . The forr Heiden  indicateAlternatively, lactate production may not be adaptive at all, but rather may reflect the fundamental metabolic constraints of eukaryotes. While some aspects of proliferation  require large amounts of ATP, overall cellular proliferation may have greater requirements for biosynthetic precursors. Many of these precursors form from glycolytic and Krebs cycle intermediates. As with any chemical reaction, an excess of product slows down or stops substrate processing. Hence, metabolizing pyruvate in the Krebs cycle and electron transport chain may produce excess ATP that may then inhibit the Krebs cycle, glycolysis, and the production of biosynthetic precursors . SimilarThese hypotheses are not mutually exclusive and likely operate together. The finding that proliferative cells use, for example, malate dismutation would have implications in this context. With malate dismutation, cytosolic NADH is reoxidized by the formation of malate from oxaloacetate . Malate The physiology of proliferation in metazoans that could reoxidize NADH via pathways other than the canonical electron transport chain or converting pyruvate into lactate ,11,12 maLife generally consists of two aspects: (1) extracting energy from the environment and converting it into useful forms and (2) using some of that energy to replicate a store of information ,23,24. UIn a broad outline, the early history of metazoans can be characterized by a dichotomy between clonal and colonial animals , and stem bilaterians. From this view, stem ctenophores were also clonal. In the context of this oversimplified but still useful dichotomy, stem bilaterians were described as “assembly-line animals” that grow rapidly, disperse widely, and seek out patches of food to consume before sexually reproducing . Clonal Drosophila. Of course, Drosophila is the archetypal assembly-line animal, virtually postmitotic as an adult and incapable of reproduction except via gametes. Morgan’s “fly room” became legendary, and much of the early history of the field of genetics was closely tied to Drosophila.In the early 20th century, a number of prominent scientists focused on the problem of regeneration, using clonal or colonial animals as their models . After 1Hydractinia symbiolongicarpus, Podocoryna carnea, and Eirene sp. have been maintained in our laboratory for decades. Periodically, with the exception of H. symbiolongicarpus, these clones produce medusae that reproduce sexually [Ideally, studies of proliferation should focus on a life-cycle stage of an organism that is capable of unlimited proliferation, at least on the basis of human time scales. Clones of colonial marine hydroids such as sexually . MeanwhiIn many ways, cnidarians, which represent an early-diverging group of metazoans , remain While physiological studies are necessary, some insight can be gained from genetic data. A key difference in the function of the aerobic compared to the anaerobic mitochondrial electron transport chain is that the former uses ubiquinone (UQ) as an electron carrier, while the latter used rhodoquinone (RQ) . In the Besides RQ itself, anaerobic mitochondria require several other genetic variants compared to aerobic mitochondria. In particular, variants of three additional genes are associated with an RQ-based anaerobic metabolism . The besC. elegans [Besides COQ-2e and Complex II, there are two other important indicators of an RQ-based metabolism . TDO-2 a elegans ,17,33. CCaenorhabditis elegans, NP_871684.1), exhibited two critical residues  that are required for RQ-based anaerobic metabolism [Acropora and Exaiptasia lack both residues, while Nematostella exhibits both, and Hydra exhibits one with a variant residue at the other position. Other corals generally exhibit aerobic character states. Anaerobic mitochondria in Nematostella may be highly beneficial, since it is found in the intertidal and shallow subtidal. Various species of Hydractinia, Podocoryna, and Eirene are also intertidal and shallow subtidal. These results suggest that Acropora, studied by Linsmayer et al. [Using four required and specific proteins for RQ metabolism ,17,33, tr et al. , has oblr et al. . On the Caenorhabditis elegans, NP_001366681.1). A single residue diagnoses the form used in anaerobic metabolism [Hydra, Nematostella, and other sea anemones, and one of the two surveyed soft corals. Hard corals , however, generally lack this residue. TDO-2 is the third protein required for RQ-based anaerobic metabolism  that exhibits one critical residue, P133, which is part of the PLD loop required for TDO-2 enzyme activity [Caenorhabditis elegans, NP_001379625.1) is also required for RQ-based metabolism [The second protein associated with RQ-based metabolism is MEV-I, a Complex II subunit  and flies (Drosophila) are essentially postmitotic as adults. On the other hand, biologists in the early 20th century had useful models to study proliferation, such as planarians and hydroids [Clytia hemisphaerica has a genomic sequence, we were unable to find any sequences related to the four proteins described here using our methods.The models of modern biology  were never conceptualized as systems to study proliferation. Rather, they were prized for exactly the opposite features—a short period of proliferation followed by a rapid initiation of the sexual phase of the life cycle. These features rendered them valuable genetic models, but less than ideal systems to study proliferation. Indeed, worms (hydroids . Those hhydroids ) might bIn hydroids with a metagenic life cycle, the proliferation of the polyp state can be controlled simply by manipulating the supply of nutrients. Hence, an experimental approach involving nutrient manipulation is suggested. If highly proliferative (nutrient-abundant) hydroids create more products of anaerobic metabolism and consume less oxygen than less proliferative (nutrient scarce) hydroids do, the central tenets of the Warburg effect are supported in metazoans that are phylogenetically distant from vertebrates. On the other hand, if these products are not primarily lactate, a role for lactate in proliferation can be deemed unlikely. Rather, the reoxidization of NADH using anaerobic pathways in proliferating cells may reflect the fundamental constraints of eukaryotic physiology, resulting in the maladaptive excretion of end products that could be used as biosynthetic precursors and thus fuel proliferation. Three related hypotheses were mentioned above to explain this putative constraint: (1) the formation of ATP in quantities that trigger end-product inhibition, shutting down the Krebs cycle and glycolysis ; (2) an Since these are marine organisms with limited homeostatic capabilities, the experimental conditions are essentially those of seawater  and ambient temperatures (roughly 20.5 °C). Given that these organisms are poorly known, there may be aspects of cellular metabolism besides those directly related to RQ-based metabolism that also show functional differences in facultative versus obligate aerobic mitochondria.Nematostella) exhibits all of the critical residues that are necessary for an RQ-based anaerobic metabolism. The simplest explanation for this pattern is that the common ancestor of cnidarians possessed facultative anaerobic mitochondria, while some modern species have lost these capabilities, but retain some of the enzymatic features, perhaps because of pleiotropy. Martin et al. [Available genomic data from cnidarian models show that all exhibit some features of facultative anaerobic mitochondria, but only one  exhibit limited cellular proliferation before initiating the sexual phase of the life cycle. Such models are hardly suitable for studies of proliferation. Other metazoans are more favorable to such studies. Colonial marine hydroids with a metagenic life cycle were highlighted here. In these hydroids, the polyp stage of the life cycle only carries out mitotic proliferation. Such a dedicated stage of the life cycle allows for proliferation to be regulated entirely by nutrient manipulation. Nutrient-abundant  and nutrient-scarce (less proliferative) treatments can be compared for physiological parameters related to the Warburg effect . This perspective allows for a clearer picture of the Warburg effect to emerge.


To propose additional items for established dry eye disease (DED) instruments that cover blepharitis-specific signs and symptoms and to determine the association between the clinical findings and subjective complaints.Thirty-one patients with blepharitis and DED were prospectively included in the pretest period for selecting suitable questions. In the main phase of the study, the selected questions were then tested on 68 patients with blepharitis and DED and 20 controls without blepharitis or DED. Pearson’s coefficient of correlation was calculated between the blepharitis-specific questions, tear break-up time (TBUT), the Schirmer test score, and the ocular surface disease index (OSDI) score; and the similarity between the blepharitis-specific questions, OSDI questions, and objective parameters for DED was assessed via hierarchical clustering. Furthermore, the discriminatory power of the blepharitis-specific questions was investigated with the receiver operating characteristic (ROC) curve.r = 0.45, p < 0.001) and Schirmer score . Cluster analysis demonstrated the similarity between the question about heavy eyelids and TBUT. In addition, the OSDI questionnaire had the highest discriminatory power in ROC analysis, and the OSDI score significantly correlated with the specific questions about eyelids sticking together  and watery or teary eyes .The additional question about heavy eyelids revealed a significant correlation with the OSDI score (The blepharitis-specific additional questions were closely associated with objective parameters for DED. The question about heavy eyelids might be well suited for recording the symptoms of hyposecretory and hyperevaporative dry eye with blepharitis. Propionibacterium acnes, Bacillus oleronius, and Demodex may cause changes in polar lipid composition and increased melting temperature in meibum, which can lead to hyperevaporative dry eye [The fundamental mechanism of dry eye disease (DED) includes tear hyperosmolarity resulting from reduced lacrimal secretion and/or a tear film lipid layer deficiency related to meibomian gland dysfunction (MGD). In MGD, the formation of lipases and esterases through proliferation of Staphylococcus,  dry eye , 2. Conv dry eye .Chronic blepharitis may affect 37–47% of all patients who visit an ophthalmologist . SeventyThe use of a questionnaire, such as the ocular surface disease index (OSDI) or Standard Patient Evaluation of Eye Dryness (SPEED), can help uncover or track symptoms related to ocular discomfort in DED patients. The OSDI has become established for wide use in clinical trials with a good specificity of 83% and a moderate sensitivity of 60%, composed of 12 questions about the frequency of DED symptoms, their impact on quality of life, and possible environmental triggers .This study aimed to suggest additional items for established DED questionnaires that quantify blepharitis-specific signs and symptoms, to correlate the blepharitis-specific questions with objective parameters for DED, and to provide new insights into the association between the clinical findings and subjective complaints in DED and blepharitis. Patient’s symptoms might be quantified with the help of blepharitis-specific questions, making it possible to accurately assess the efficacy of a therapy and establish therapy monitoring.In order to pick appropriate questions in the main phase of the study, we prospectively enrolled 31 patients with concurrent blepharitis and DED who had answered 43 different blepharitis-associated questions at the Eye Center of Freiburg University between 2019 and 2020 in the pretest phase. The seven selected questions from the pretest phase were then assessed on 68 patients with blepharitis and DED in the study’s main phase. Blepharitis, characterized by inflammation of the eyelids, can be diagnosed by symptoms and signs, e.g., redness, irritation, burning, tearing, itching, crusting of eyelashes, loss of eyelashes, eyelid sticking, blurring or fluctuating vision and photophobia along with the measurement of visual acuity, an external examination of skin and eyelids, slit-lamp biomicroscopy including the evaluation of eyelid margin, eyelashes, conjunctiva, cornea, tear meniscus, tear film break-up time, and pattern. Twenty patients’ companions without blepharitis or DED were employed as healthy controls free of eye diseases to test the specificity of additional questions, who might have similar environmental factors and family history to the patients. Clinical data, such as age at diagnosis, sex, vision, slit lamp exam results, tear break up time (TBUT), Schirmer test score with anesthesia, questionnaire responses and photo documentation, were collected. The exclusion criteria were ocular surface diseases other than DED, e.g., atopic keratoconjunctivitis, herpetic keratoconjunctivitis, ocular pemphigoid, and ocular graft-versus-host disease (GVHD). This study was approved by the Ethics Committee at the University of Freiburg . An anonymous analysis was conducted in accordance with local ethical guidelines.In the pretest phase, patients were asked about the intensity with a visual analogue scale (VAS) and the frequency of the following symptoms: gritty eyes, dry eyes, itchy eyes, heavy lids, eye/eyelid burning, eyes sensitive to light, tired eyes, blurred vision, watery or teary eyes, crusting/flakes on the eyelids, eyelids sticking together , swollen lid margins, loss of eyelashes, and eyelid redness. Patients needed to answer how often the symptom occurred during the last week by selecting the appropriate option from “All of the time,” “Most of the time,” “Half of the time,” “Some of the time,” and “None of the time.” Additionally, questions about the limitations of reading, driving at night, using the computer, watching TV, sleeping, using a mobile phone, and wearing contact lenses were included. Finally, patients were asked if they were generally most bothered by how their eyes look or feel.By analyzing the correlation of the questions and the distribution of the answers, the questions about teary eyes, heavy eyelids, crusts/dandruff on the eyelids/eyelashes, sticking eyelids, swollen eyelid margins, and red eyelid margins were selected as the appropriate additional question in the main phase Fig. . The queThe most widely used test of tear film stability in clinical practice has been the TBUT test . A stripTo select appropriate questions for the main phase of the study, correlation heat map of 43 blepharitis-associated questions and clinical parameters in the pretest phase were generated, and distribution of answers to all questions was compared. For descriptive statistical analysis, median values for age at diagnosis, vision, TBUT, the Schirmer test score, and the OSDI score recorded in the study’s main phase were calculated. To examine association between the clinical findings and subjective complaints, Pearson’s coefficient of correlation between the blepharitis-specific questions and clinical parameters for DED was calculated, followed by hierarchical clustering and an analysis method using a generalized linear model. Unless otherwise stated, the ophthalmological parameters of the right eye were selected for quantitative analysis in the main phase because there was a strong positive correlation between the clinical data of the right and left eye. Furthermore, we compared the discriminatory power of the blepharitis-specific questions and the OSDI with the receiver operating characteristic (ROC) curve. Moreover, patients were divided into two groups according to sex, and the sex-specific differences in symptoms were investigated by comparing the distribution of answers. All calculations were performed using the R-System . A significance level was set at 0.05. We did not adjust for multiple testing due to the explanatory character of our investigation.Sixty-eight patients with blepharitis and DED and 20 controls without blepharitis or DED were included in the main phase of the study. The characteristics of the patients and the control group are summarized in Table p value were determined and demonstrated in Table r = 0.47, p < 0.0001), heavy eyelids , and tearing . In addition, a significant negative correlation was found between the Schirmer test score and heavy eyelids , indicating a possible association between heavy eyelids and hyposecretory dry eye. However, no significant correlation was observed between TBUT and the blepharitis-specific additional questions.A bivariate correlation analysis of three blepharitis-specific additional questions about eyelids sticking together, heavy eyelids, and tearing, the OSDI score, TBUT, as well as the Schirmer test score with violin plot visualization Fig.  were perCluster analysis visualized with dendrogram revealed similarity structures for all blepharitis-specific additional questions in the main phase, the OSDI questions, TBUT, and the Schirmer test score Fig. . Figure In the ROC analysis, the “hit rate” referred to the proportion of the patient group identified by the question in the questionnaire, i.e., the percentage of patients in the patient group that had the symptom. The “false alarm rate” described the percentage of the control group that the questionnaire question detected, namely, the proportion of the controls with the symptom in the control group. The 12 OSDI questions had the highest discriminatory power between the controls without DED or blepharitis and the patients with DED and blepharitis, outperforming the additional questions about eyelids sticking together, heavy eyelids, crust or flakes on eyelashes, and swollen eyelid margins Fig. . Moreovep = 0.006), tearing , heavy eyelids , redness at the eyelid margins , and crust or flakes on eyelashes , demonstrating that the OSDI score significantly correlated with these blepharitis-specific additional questions. Additionally, the Schirmer test score was a significant factor in the models for the questions about heavy eyelids  and swollen eyelid margins .A generalized linear model was used to test whether the results of all blepharitis-specific additional questions in the main phase could be predicted by the OSDI score, the Schirmer test score, and TBUT. The test results revealed that the OSDI score was a significant factor in the models for the questions about eyelids sticking together . In addition, 20% of women chose the option “All of the time” for the OSDI question about gritty eyes, while no men responded with the same answer (p = 0.039). Overall, these results indicated that women had stronger subjective perceptions of gritty eyes and feeling uncomfortable in very dry places than men.Significant sex differences in the distribution of answers were compared. Thirty-one percent of female patients answered “Most of the time” to the OSDI question about discomfort in places with low humidity, whereas 11% of male patients gave the same answer  and the Schirmer test score  and the same cluster membership as that of TBUT in cluster analysis. The similarity between the question about heavy eyelid and TBUT in cluster analysis may be explained by the fact that meibomian gland dysfunction can lead to the tear film instability, increased evaporation and eventually a hyperevaporative dry eye. Taken together, we concluded that the question about heavy eyelids could be used to assess symptoms of both hyposecretory dry eye and hyperevaporative dry eye, while the OSDI was more applicable to deficiency of the aqueous-mucinous tear film components due to lack of correlation with the parameters for diagnosing hyperevaporative dry eye [This study demonstrates that the blepharitis-specific additional questions can assess the symptoms of patients with blepharitis and DED, when significantly correlated with the clinical parameters for DED. Our results are in line with a psychometric study presenting a significant strong correlation between the OSDI and blepharitis-associated symptoms, which included heavy or swollen eyelids, redness, watery eyes, crusty or scaly eyelids, and eyelids sticking together . Among t dry eye . The coep = 0.015) and gritty eyes (p = 0.039). This observation is in accord with another study suggesting that women with DED are more likely than men to experience symptoms such as feeling uncomfortable with environmental triggers [Furthermore, a significant sex-specific difference was determined in the distribution of answers to the OSDI questions about discomfort in places with low humidity  with a specific collective of computer users. The discrepancy between clinical signs and symptoms in DED is consistent with the notion that sensitization of the ocular sensory pathways may be triggered by events occurring long before the patient visits the ophthalmic clinic [The frequent coexistence of DED and blepharitis allowed for the prospective character of the current study with good data quality and high power. Despite the limited number of study participants, we detected a significant correlation between the blepharitis-specific additional questions and objective clinical parameters. However, no significant correlation between TBUT and the blepharitis-specific additional questions was noted in this study. Finis et al.  also fouc clinic –19. Whenc clinic .In the ROC analysis, the OSDI score (or the 12 OSDI questions?) had the highest discriminatory power in distinguishing the control group from the patient group, outshining any blepharitis-specific additional questions. This may be attributed to the fact that most of the controls in the main phase of the study did not suffer from DED, so that the blepharitis-specific additional questions could not show any difference. Future studies could attempt to include a group of patients with DED and without blepharitis to test the discriminatory power of the blepharitis-specific additional questions. The findings might be impacted by age and sex disparities between the patients and healthy controls, as old age and female sex are risk factors linked to DED.In conclusion, the present study exhibits significant correlations between the clinical parameters for DED and the blepharitis-specific additional questions for assessing blepharitis in DED. The question about heavy eyelids may be best suited to describe the symptoms of hyposecretory and hyperevaporative dry eye with blepharitis due to significant correlations with the OSDI score and the Schirmer test score, as well as the same cluster membership as from TBUT in cluster analysis. In addition, this study demonstrated that women with DED and blepharitis had significantly stronger subjective perceptions of gritty eyes and discomfort than men in places with low humidity.There are limitations to this study. With a small study pool, caution must be applied, as the results might be lack of reliability due to insufficient range of responses. It is necessary to prove the specificity of the additional questions for blepharitis with quantitative measurements of meibomian gland dysfunction such as lipid layer thickness by LipiView interferometer. As fluorescein makes the tear film less stable, the measurement of TBUT may not be an accurate representation of its condition. Spearman’s correlation coefficient and internal consistency reliability, often measured using Cronbach’s alpha, should be calculated for the values with non-normal distributions.The significant correlations between the objective parameters for dry eye disease und the questions about signs and symptoms of blepharitis reflected the close relationship between dry eye disease and blepharitis. Case–control studies, prospective cohort studies, molecular genetic analysis, and bioinformatics analysis will be required to investigate the relationship between dry eye disease and blepharitis.


Graphene is an emerging nanomaterial increasingly being used in electrochemical biosensing applications owing to its high surface area, excellent conductivity, ease of functionalization, and superior electrocatalytic properties compared to other carbon-based electrodes and nanomaterials, enabling faster electron transfer kinetics and higher sensitivity. Graphene electrochemical biosensors may have the potential to enable the rapid, sensitive, and low-cost detection of cancer biomarkers. This paper reviews early-stage research and proof-of-concept studies on the development of graphene electrochemical biosensors for potential future cancer diagnostic applications. Various graphene synthesis methods are outlined along with common functionalization approaches using polymers, biomolecules, nanomaterials, and synthetic chemistry to facilitate the immobilization of recognition elements and improve performance. Major sensor configurations including graphene field-effect transistors, graphene modified electrodes and nanocomposites, and 3D graphene networks are highlighted along with their principles of operation, advantages, and biosensing capabilities. Strategies for the immobilization of biorecognition elements like antibodies, aptamers, peptides, and DNA/RNA probes onto graphene platforms to impart target specificity are summarized. The use of nanomaterial labels, hybrid nanocomposites with graphene, and chemical modification for signal enhancement are also discussed. Examples are provided to illustrate applications for the sensitive electrochemical detection of a broad range of cancer biomarkers including proteins, circulating tumor cells, DNA mutations, non-coding RNAs like miRNA, metabolites, and glycoproteins. Current challenges and future opportunities are elucidated to guide ongoing efforts towards transitioning graphene biosensors from promising research lab tools into mainstream clinical practice. Continued research addressing issues with reproducibility, stability, selectivity, integration, clinical validation, and regulatory approval could enable wider adoption. Overall, graphene electrochemical biosensors present powerful and versatile platforms for cancer diagnosis at the point of care. Cancer remains one of the leading causes of mortality worldwide, responsible for nearly 10 million deaths in 2020 alone 3−/4− probe. The Au NPs contribute to the high sensitivity of the immunosensor. Bharti et al. [Depositing noble metals like gold and platinum generates nanocomposites with excellent electrocatalysis and high surface area . Elshafei et al.  reported−14 M with a linear range spanning from 1.0 × 10−14 to 1.0 × 10−7 M. Clinical trials successfully detected CYFRA21-1 DNA in real lung cancer samples, showcasing its potential for early lung cancer diagnosis.Three-dimensional graphene foams, sponges, and hydrogels provide enormous surface area for biofunctionalization. Crosslinking GO with small molecules or polymers produces porous networks ideal for loading high quantities of nanomaterials and biomolecules. Su et al.  describeOverall, the diversity of oxygen groups on GO coupled with the ability for metal doping and 3D assembly allow for extensive modifications to improve graphene’s performance in electrochemical biosensing.Various designs for electrochemical biosensors that incorporate graphene materials as the transducer have been explored for sensitive and selective cancer diagnosis. These utilize different sensor fabrication strategies including GFET, graphene-modified electrodes, nanocomposites with graphene, and 3D graphene networks. Each configuration offers specific advantages arising from graphene’s unique properties. This section highlights these major graphene electrochemical biosensor platforms along with their preparation, operating principles, and performance advantages.Graphene is an ideal transducer material for FETs due to its exceptional charge carrier mobilities allowing rapid current modulation . Graphen2. For example, a sensor may use CVD-grown graphene as the transducing element in a FET biosensor for detecting the cancer biomarker HER3 [Graphene can be incorporated into FETs through various fabrication strategies. Large-area graphene synthesized by CVD allows wafer-scale production but requires transfer onto insulating substrates like SiOker HER3 . The gra2 substrate, forming the basis of the sensor  with an amino group at the 5’ end is immobilized directly onto a graphene-modified GCE through π–π stacking interactions between the DNA bases and graphene surface [2 to 1.12 × 108 particles/μL, with a limit of detection of 96 particles/μL. The key advantages of this biosensor were the use of click chemistry for the efficient nonspecific labeling of exosomes and HCR signal amplification to enhance sensitivity.Immobilized single-stranded DNA or RNA probes on graphene transduce hybridization events with complementary nucleic acid targets into measurable electrical signals for sensitive and selective cancer diagnosis. Physical adsorption based on π–π stacking between nucleotide bases and graphene sp surface . The tar surface . It enab surface  reportedElectrostatic attraction aids in the immobilization of negatively charged DNA but requires positively charged surfaces. Pyrene , poly-L-Graphene-based electrochemical biosensors have been widely explored for the sensitive and selective detection of a variety of cancer biomarkers, including proteins, DNA, CTCs, miRNAs, and metabolites. Early diagnosis and screening to identify these biomarkers can significantly improve clinical outcomes for cancer patients. This section highlights the various applications of graphene electrochemical sensors for the analysis of different cancer biomarkers.A number of protein biomarkers are extensively used in cancer diagnosis, prognosis, and the evaluation of treatment efficacy. PSA is the most commonly used biomarker for early prostate cancer screening. Meng et al.  reportedCEA is an important clinical biomarker for colon, breast, lung, and pancreatic cancer diagnosis and disease monitoring. A new electrochemical immunosensor for the detection of CEA was reported by . The imm−11 mg/mL, with a wide linear detection range of 10−10 to 10−4 mg/mL OVA. Klukova et al. [In addition to protein biomarkers, aberrant glycan patterns serve as signatures for cancer progression and metastasis. Graphene-oxide-based electrochemical biosensors have been developed for detecting cancer-associated glycoproteins. Huang et al.  reporteda et al.  describeCTCs are cancer cells shed from solid tumors into the bloodstream, serving as an emerging biomarker for cancer diagnosis and prognosis. The isolation and detection of rare CTCs among billions of blood cells remains technically challenging. GO substrates have been explored with regard to the sensitive capture and electrochemical detection of CTCs, leveraging the enhanced adhesion of CTCs onto graphene surfaces . The apt−15 to 1 × 10−7 M BRCA1, with a very low limit of detection of 3.01 × 10−16 M.Nucleic acid biomarkers like mutations, single nucleotide polymorphisms, altered gene expression, and methylation patterns provide valuable tools for cancer diagnosis. Cai et al.  fabricatThe TP53 gene, also known as the tumor protein 53 or p53, is one of the most well-known tumor suppressor genes in the human body. Its role is critical in preventing the development and progression of cancer. Mutations in the TP53 gene are implicated in a wide range of cancers, and its importance in cancer diagnosis and understanding is substantial. Avila et al.  develope−4 μg/mL for detecting KRAS. The sensor with azulene dye A1 and TiO2-Pt/reduced graphene oxide had a lower limit of quantification of 2.64 × 10−7 μg/mL KRAS. These sensors were selective for KRAS with minimal interference from other substances. The sensors were used to detect KRAS in whole blood samples from colon cancer patients and results correlated well with KRAS detection in tumor tissue samples. The electrochemical sensors show promise as sensitive and selective tools for the minimally invasive detection of the colon cancer biomarker KRAS. Shu et al. [The KRAS gene  is a critical gene that codes for a protein involved in cell signaling pathways. Mutations in the KRAS gene are commonly associated with various types of cancers, particularly colorectal, lung, and pancreatic cancers. The presence of KRAS mutations is important for cancer diagnosis and treatment. Al-Ogaidi et al.  used modu et al.  fabricatu et al.  reported2. Testing in spiked blood samples showed good selectivity and recovery. MicroRNAs are short, non-coding RNAs that regulate gene expression and serve as promising biomarkers for cancer diagnosis and prognosis. Pothipor et al.  designedWhile graphene electrochemical biosensors show potential for sensitive and selective cancer diagnosis, there remain significant challenges to translating this emerging technology into widespread clinical adoption. Ongoing research efforts aim to address these challenges and open up new opportunities to further improve analytical performance and real-world applicability.One key challenge is regarding problems with the reproducibility and reliability of sensor fabrication and performance. Graphene synthesis methods, functionalization strategies, immobilization chemistries, and nanomaterial assembly introduce variability in the final sensor architecture and behavior. This leads to sensor-to-sensor disparities in sensitivity, selectivity, linear range, and limits of detection during cancer biomarker analysis. Establishing standardized fabrication protocols and quality control measures will be essential. Automation using microfluidics and lab-on-a-chip approaches could aid large-scale manufacturing. The systematic optimization of surface functionalization and nanomaterial integration can tailor sensor designs for specific cancer biomarkers to improve reliability across sensor batches.Another major obstacle is the poor stability and shelf life of current graphene electrochemical biosensors. Biomolecule immobilization and the subsequent target binding introduce conformational changes in receptors like antibodies and aptamers over time, reducing their binding affinity. The numerous nanomaterial interfaces are also susceptible to degradation. This impacts the long-term storage ability and reproducibility of sensors. Stabilization strategies including crosslinking approaches for biorecognition elements and the addition of protective polymers or surfactants for nanomaterials need investigation. Developing calibration and blocking protocols to mitigate activity loss prior to sensor use would enhance robustness.Selectivity against the complex milieu of non-specific proteins, biomarkers, and cells present in real patient samples poses a further key challenge. Novel selective membranes, hydrogels, and polymers that prevent fouling while allowing for the diffusion of specific targets to the sensor require exploration. Graphene itself can be doped with atoms like nitrogen to improve its hydrophilicity and antifouling properties. Advanced surface functionalization for greater target specificity, combined with optimized sensor geometries maximizing target capture, can enhance selectivity. The use of auxiliary reagents and data processing to filter non-specific signals can augment performance in biological fluids. Expanding testing with diverse patient samples rather than spiked artificial samples will clarify clinical viability.Most reported electrochemical biosensors use planar 2D graphene electrodes, which limit sensitivity due to their modest surface area. Scalability also remains constrained. Moving to 3D porous graphene networks, composites, and foams provides greater surface area for enhanced biomarker capture and signal transduction. However, optimizing synthesis conditions for reproducible 3D nanostructures and preventing biomolecule leaching from the pores requires work. Free-standing 3D graphene materials allow ease of integration with existing electrode substrates. Mass producible designs leveraging screen printing show promise for commercial translation.Integration with microfluidics and lab-on-a-chip technology can allow automated sample processing, biomolecule separation, target preconcentration and multistep analysis workflows to further augment graphene sensor performance and practicality. Multiplexing capabilities can be enhanced by fabricating sensor arrays tailored to different cancer biomarkers. Coupling with smartphone connectivity and readout circuits will enable point-of-care operation. Patient studies to validate clinical accuracy against established methods like ELISA and PCR in identifying early-stage cancers and monitoring treatment are vital. Regulatory approval will be contingent on demonstrations of reliability, safety, efficacy, and health outcomes relative to standard of care. An emerging opportunity is the integration of graphene with well-established silicon-based semiconductor technologies for leveraging their complementary advantages. For example, graphene can be integrated with silicon FETs through techniques like the CVD growth of graphene on silicon. This allows graphene’s high sensitivity to be combined with silicon’s ease of manufacturing and integration with electronics. Hybrid graphene-silicon biosensors could potentially offer scalable fabrication along with the high performance of graphene for point-of-care diagnostic devices.Expanding the breadth of targets beyond common protein antigens and DNA mutations presents a rich opportunity area. The graphene platform offers versatility for the detection of many emerging cancer biomarkers like exosomes, circulating tumor cells, metabolites, and glycoproteins. Explorations into novel biorecognition elements beyond antibodies and DNA probes can impart greater affinity, specificity, and multiplexing capabilities. The hybridization of biological molecules with synthetic reagents through bioconjugation chemistries can augment performance.Harnessing the full scope of electrochemical techniques beyond amperometry, such as impedimetric, potentiometric, and conductometric transduction, can reveal deeper insights into target binding mechanisms and cancer states. The modification of electrode interfaces with novel nanomaterials to impart enzyme-mimicking properties for enhanced electrocatalysis warrants study. Extending lab-based research to animal models and clinical settings will accelerate translation towards practical cancer diagnostics, theranostics, and point-of-care testing.Extensive clinical validation with large patient sample sizes is essential to establish sensitivity and selectivity in real patient samples, which may behave differently from spiked samples in buffer solutions. Stringent quality control and testing is critical prior to regulatory approval for clinical use. Furthermore, toxicity concerns with certain forms of graphene need to be addressed to ensure biocompatibility.Realizing the immense potential of graphene electrochemical biosensors for sensitive, rapid, and low-cost cancer diagnosis faces ongoing challenges in reproducibility, stability, selectivity, scalability, integration, validation, and commercialization. However concerted research efforts to address these challenges and expand capabilities promise continued exciting progress, accelerating their clinical implementation. Ongoing developments in advanced nanomaterial design, surface functionalization, electrochemical techniques, microfluidic integration, and biorecognition element innovation in conjunction with expanded clinical testing will usher these flexible and powerful platforms into mainstream use for early cancer detection, guiding therapeutic interventions and improving patient outcomes.In summary, graphene-based electrochemical biosensors have emerged as promising platforms for the sensitive, selective, and rapid diagnosis of cancer. The exceptional properties of graphene, including high surface area, excellent conductivity, ease of functionalization, and superior electrocatalytic activity, enable the construction of sensors with improved performance compared to traditional electrode materials. The ability to efficiently immobilize a variety of biorecognition elements like antibodies, aptamers, peptides, and DNA/RNA probes imparts specificity towards cancer biomarkers. Additionally, graphene composites with nanomaterials, 3D porous architectures, and microfluidic integration further augment analytical capabilities and practical functionality.Significant advances have occurred in developing graphene electrochemical biosensors for detecting key cancer biomarkers such as proteins, circulating tumor cells, DNA mutations, miRNAs, and metabolites. Innovative sensor designs, immobilization strategies, and signal enhancement approaches have enabled detection limits down to femtomolar and even attomolar levels, alongside high selectivity. Testing with clinical samples shows promise for early diagnosis and point-of-care assessment. However, challenges remain including reliability, reproducibility, stability, scaling up, selectivity in complex media, system integration, validation, and regulatory approval.Ongoing research to address these challenges, while expanding the range of detectable biomarkers and electrochemical techniques, continues to push graphene biosensor technology forward. Their flexibility, sensitivity, simplicity, and affordability make them well-suited for transitioning cancer diagnostics from centralized labs towards widespread point-of-care clinical use. The standardization of fabrication protocols and quality control measures will aid manufacture. The optimization of surface chemistries, advanced nanocomposite materials, 3D architectures, microfluidic integration, and expanded multiplexing capabilities can further improve performance and practicality. Wider clinical testing and demonstrations of health outcomes versus standard methods are critical to regulatory approval and healthcare adoption. Overall, graphene electrochemical biosensors promise continued exciting advances in coming years, helping to enable early diagnosis, cancer screening, and personalized therapy monitoring to improve patient outcomes.


Mycobacterium avium subsp. paratuberculosis (MAP) as a cause of sarcoidosis. MAP is involved in a parallel story as the putative cause of Crohn’s disease, another disease featuring noncaseating granulomas. MAP is a zoonotic agent infecting ruminant animals and is found in dairy products and in environmental contamination of water and air. Despite increasing evidence tying MAP to several human diseases, there is a continued resistance to embracing its pleiotropic roles. “Who Moved My Cheese” is a simple yet powerful book that explores the ways in which individuals react to change. Extending the metaphor, the “non-cheesy” granuloma of sarcoidosis actually contains the difficult-to-detect “cheese”, MAP; MAP did not move, it was there all along.Clinical and histological similarities between sarcoidosis and tuberculosis have driven repeated investigations looking for a mycobacterial cause of sarcoidosis. Over 50 years ago, “anonymous mycobacteria” were suggested to have a role in the etiology of sarcoidosis. Both tuberculosis and sarcoidosis have a predilection for lung involvement, though each can be found in any area of the body. A key histopathologic feature of both sarcoidosis and tuberculosis is the granuloma—while the tuberculous caseating granuloma has an area of caseous necrosis with a cheesy consistency; the non-caseating granuloma of sarcoidosis does not have this feature. This article reviews and reiterates the complicity of the infectious agent,  The first clinical record of sarcoidosis dates to 1859 when Jonathan Hutchinson of the Blackfriers Hospital for Diseases of the Skin of London described a patient with raised, purple skin plaques; initially Hutchinson related them to the patient’s gout. Upon seeing another individual, a Mrs. Mortimer, with similar dermatologic findings, Hutchinson differentiated the lesions from tuberculosis and lupus and labeled the condition “Mortimer’s malady” . PulmonaMycobacterium avium subsp. paratuberculosis (MAP), or a closely related M. avium complex [MAC] strain, from sarcoid skin lesions and cerebrospinal fluid [Common to many inflammatory/autoimmune diseases is the concept of an “environmental trigger.” Infectious agents have been the suspected cause of sarcoidosis since the early 1900’s with mycobacteria considered the likely agent ,6,7. In al fluid .A hallmark of sarcoidosis is the non-caseating granuloma . HoweverM. tuberculosis produces a virulence factor, trehalose 6,6′ dimycolate, that is markedly toxic [M. tuberculosis antigens [Caseous necrosis is associated with tuberculosis; ly toxic ; it is tly toxic . While iantigens . Howeverantigens   suggesting a no-longer-anonymous mycobacterium in sarcoidosis but identifying a “mycobacterium of interest” as MAP [Caseating and non-caseating granulomas are types of inflammatory lesions. Caseating granulomas are characterized by the presence of dead or necrotic tissue that has a coidosis . Another” as MAP . MAP belongs to the MAC category that comprises over 10 species and subspecies, some of which are pathogenic to animals and humans . MAP is Inefficiency of diagnostic testing for MAP along with the long latency of infection in apparent uninfected yet productive dairy animals create hesitancy by producers to test their herds; moreover, there is no mandate requiring testing. Collectively, this results in more MAP-shedding animals that propagate infection and contaminate milk and meat, promotes trade of asymptomatic animals, and delays the mainstay by which Johne’s disease is controlled, animal culling .MAP is a resilient organism and shedding by infected animals is a major source of environmental MAP; once excreted, MAP can survive up to 120 weeks in soil or water . MAP is Both Iceland and what is now the Czech Republic were historically isolated from Johne’s disease due to limited importing/exporting of ruminant animals. For Iceland this changed in 1933 when apparently healthy, but sub-clinically MAP-infected sheep were imported . Other sM. leprae, the microbial cause of leprosy that has never been grown in culture, MAP is the slowest growing of the mycobacteria with some labs continuing for a year before calling a culture MAP-negative. Veterinary laboratories employing both enhanced conventional and molecular methods for identifying MAP in ruminant animals such as cattle and sheep have the greatest experience in detecting MAP [Regarding human Crohn’s samples, the basis of the hundred-year controversy is the fact that traditional culturing and staining are largely unsuccessful in identifying MAP ,32,33. Ating MAP . In addition to being difficult to culture, MAP can exist with a modified cell wall—the component of the mycobacterium that takes up a characteristic acid-fast stain. MAP can shed its cell wall, becoming a spheroplast or L-form . The bacHumans are exposed to MAP in contaminated food, water, and air . A primaFor this review, MAP in cheese is featured. The U.S. Food and Drug Administration allows two options for producing safe cheese products: either use pasteurized milk in cheese production or cure the finished cheese for at least 60 days at temperatures of 2 °C. The curing process can include low pH and various salt concentrations to lessen pathogens; like pasteurization, efforts in the curing process to curtail MAP have some, but not total success, with MAP in cheeses . MAP is To date, MAP has been associated with Crohn’s disease, Blau syndrome, autoimmune (Hashimoto’s) thyroiditis, autoimmune diabetes, multiple sclerosis, lupus erythematosus, rheumatoid arthritis, and Parkinson’s disease ,53. WhilMachine learning as applied to the microbiome is a nascent science for developing biomarkers of specific disease states. It has recently been applied in cattle to generate a microbiota signature for MAP infection . In a spReports on the epidemiology of sarcoidosis are quite varied. Such disparity is likely due to a number of factors including ,60: (i) 5 in 1995 to 330/105 in 2010. When they examined the prevalence of sarcoidosis in Columbus, Ohio (Franklin County)—a region with a demographic profile  that is nearly identical to the U.S. as a whole—the prevalence of sarcoidosis was estimated to be 48/105 in 2010 (and may be as high as 200/105), significantly higher than the U.S. The authors speculated that although an actual increase in the number of sarcoidosis cases could be occurring over this 15-year period, greater use of CT scans, the availability of less invasive diagnostic techniques, and perhaps increased awareness of sarcoidosis by clinicians likely played a role in the increased prevalence.Erdal and co-workers  analyzed5 in 2009 to 0.97/105 in 2015 [5 in females and 7.30/105 in males (combined = 9.37/105).In a similar study from Korea of sarcoidosis between 2007 and 2016—coded by the Korean Classification of Disease—the annual incidence rates of sarcoidosis increased from 0.85/10 in 2015 . The hig5 in 1996 to 143/105 in 2015, a highly significant increase of 116% [5 in 1996 to 6.8/105 in 2014; when the incidence was stratified by gender, there was a 30% decrease in the incidence in females, but a 5.5% increase in males. The authors speculated that the higher sarcoidosis incidence in males in the latter years examined was due to an increase in occupational dust exposures as there was a significant increase in the construction industry in Ontario during this period.In a study from Ontario, Canada, the prevalence of sarcoidosis increased from 66/10 of 116% . Interes5 person-years and the annual prevalence from 79 to 141 cases/105 persons [Seedahmed and co-workers  analyzed persons . They alIn summary, while numerous studies report increasing prevalence of sarcoidosis in specific countries, it is not known whether this increased prevalence is due to increasing new incident cases , greater detection of asymptomatic and symptomatic sarcoidosis with use of CT scans, reporting bias of positive epidemiologic data, inadvertent bias due to selective inclusion of specific racial groups in whom sarcoidosis appear to be more common, or combinations of any of these elements. Even within a country, there are reports of geographic variation of sarcoidosis likely related, in large part, to differences in ethnic group populations studied ,66,67. TSarcoidosis is most likely the result of some form of genetic susceptibility plus an inciting environmental factor. There is a 3.7-fold increase in sarcoidosis with having at least one first degree relative with sarcoidosis . While sSLC11A1 gene [SLC11A1 gene is located on chromosome 2q35. It encodes an integral membrane protein of 550 amino acids that is expressed exclusively in the lysosomal compartment of monocytes and macrophages [SLC11A1 gene modulates the cellular environment in response to activation by intracellular pathogens by acidifying the phagosome thus killing the pathogen [SLC11A1 impairs phagosome acidification yielding a permissive environment for the persistence of intracellular bacteria [Risk of sarcoidosis is associated with polymorphisms of the 1A1 gene . What warophages . The propathogen . As suchpathogen . Mutatiobacteria .SLC11A1 gene [SLC11A1 defect mouse [Susceptibility to mycobacterial diseases such as tuberculosis, leprosy, and Buruli’s ulcer, are associated with polymorphism of the 1A1 gene ,76,77,781A1 gene , goats [1A1 gene , and shect mouse .SLC11A1 and sarcoidosis, polymorphisms of this gene are also found in rheumatoid arthritis [In addition to rthritis , inflammrthritis ,85, multrthritis , and autrthritis . Each ofrthritis ,53.The human leukocyte antigen (HLA) system coordinates the adaptive immune response to foreign antigens, which includes both infectious and non-infectious agents. The HLA genes encode major histocompatibility complex (MHC) molecules, which are found on antigen presenting cells that process and present antigens in the context of MHC molecules to T-lymphocytes, resulting in the initiation of the adaptive immune response to that antigen . The T-lymphocytes that proliferate in sarcoidosis often show a restricted repertoire of T-cell receptors suggesting a limited number of peptides are responsible for the expansion of these T-lymphocytes . ResearcMany HLA alleles are thought to be associated with sarcoidosis. The U.S. ACCESS study found that HLADRB1 * 1101, DRB1 * 1201, DRB1 * 1501, and DRB1 * 0402 alleles were strongly associated with sarcoidosis . AdditioHLA alleles are associated with different risks for tuberculosis as well. HLA-DRB1 * 04 and HLA-DRB1 * 08 genotypes seem to increase the risk for infection, whereas HLA-DRB1 * 03 and HLA-DRB1 * 07 genotypes seem to decrease the risk . It shouMycobacterium tuberculosis infections suggest a possibly common pathogenic pathway between sarcoidosis and mycobacterial infections. As mentioned before, HLA-DRB1 * 1101 has been associated with susceptibility to sarcoidosis. Oswald-Richter et al. demonstrated that individuals with HLA-DRB1 * 1101 were statistically more likely to recognize ESAT-6, a peptide derived from mycobacteria [H1 polarized response from sarcoidosis T-cells [H1 response is theorized to be the immune dysfunction responsible for sarcoidosis. Another study, using a bioinformatic approach, demonstrated that individuals with sarcoidosis tended to express HLA-DR allele combinations that recognize M. tuberculosis and M. avium epitopes with greater affinity than those individuals with tuberculosis, suggesting that sarcoidosis may be a hypersensitive reaction to mycobacterial antigens [Shared HLA alleles in individuals with sarcoidosis and bacteria . These a T-cells . Interesantigens . These rM. tuberculosis infections may contribute to different sub-phenotypes in both diseases. In a study by Dawkin et al., it was found that certain HLA alleles were found in higher frequencies in individuals who never developed M. tuberculosis infections despite close contact with an index case and in those with acute/resolved sarcoidosis compared to individuals with M. tuberculosis infections and chronic sarcoidosis, respectively [HLA polymorphisms in individuals with sarcoidosis and ectively .et al. found that using HLA-DRB1 alleles in combination with M. tuberculosis status enabled the authors to predict the sarcoidosis phenotype: sarcoidosis cases with either HLA-DRB1 * 03 or -DRB1 * 04 allele and the absence of M. tuberculosis DNA were associated with acute/resolved disease, while cases with HLA-DRB1 * 11, -DRB1 * 15, and the presence of M. tuberculosis DNA were associated with chronic sarcoidosis [M. tuberculosis could be an antigen that produces a more severe clinical course in sarcoidosis with certain HLA alleles [M. tuberculosis and M. avium epitopes, whereas those with chronic versions of the disease had HLA alleles with lower affinity to the same epitopes [The different phenotypes of sarcoidosis may be the result of how different HLA alleles respond to mycobacterial peptides. Grosser coidosis . The aut alleles . The stuepitopes . The autepitopes . CARD15 gene is part of the ancestral innate immune system that encodes for a cytoplasmic receptor that senses and eliminates bacteria [NOD2, it is now known as the CARD15 gene [CARD15 gene [The bacteria ; originaD15 gene . Blau syD15 gene ,101. CARD15 associated with Blau syndrome susceptibility is found within the nucleotide binding site domain [The component of e domain , wherease domain . In a sme domain . Early oe domain ,106.Cardiac involvement is an increasingly recognized and serious manifestation of sarcoidosis ranging from silent myocardial granulomas leading to sudden death ,108,109 Case Study: Cardiac sarcoidosis resolved with Mycobacterium avium subsp. paratuberculosis antibiotics (MAP) [https://otakaropathways.co.nz (accessed on 3 March 2023)). His blood was positive for MAP. The MAP-targeting anti-mycobacterial therapy [A particularly instructional sarcoidosis case was reported in 2018: cs (MAP) . The pat therapy  resulted therapy . This pa therapy ,116,117.M. tuberculosis [Although a mainstay of sarcoidosis treatment, corticosteroids are palliative but not curative with potentially major side effects . Up to 7rculosis . Variatirculosis . Likewisrculosis . An entrenched tenant is that sarcoidosis is a disease of undetermined etiology. While the parallel non-granulomatous story of MAP and Crohn’s disease has garnered an increasingly tighter association, a MAP/sarcoidosis association remains to be made known; this article proposes a causal role for MAP in sarcoidosis. As presented in various sections of this paper, MAP has been found in juvenile sarcoidosis tissues and anti-MAP antibiotic therapy has successfully treated cardiac sarcoidosis. Revealed in the epidemiology section of this article, sarcoidosis is increasing in its prevalence; also, the genetics section of this article indicates that the risks for sarcoidosis are shared risks for mycobacterial infection. Parsimony favors a causal role for mycobacteria in sarcoidosis. A mycobacterium to which humans are regularly exposed is MAP, a mycobacterium that is difficult to detect. A consensus article calling for MAP zoonotic designation was the product of a 2017 international assembly of MAP researchers—such precautionary measures have yet to be taken .The book “Who Moved My Cheese?” by Spencer Johnson, M.D. is about four characters who live in a maze and rely on finding cheese to survive . The cheThis article, like the book, asks the reader to consider something unfamiliar: the inertia of MAP’s causal involvement in an increasing list of inflammatory and autoimmune diseases as it applies to sarcoidosis . UnderstIn the book, characters searching the maze for cheese leave messages on the wall—they do this in hopes that those who follow will “see the writing on the wall.” Extending the metaphor, the writing on the wall of this article is that although there is no apparent cheesy necrosis in the noncaseous granulomas of sarcoidosis, the sought-after cheese (“MAP”) was there all along.


When graphene, which exhibits both superhigh TC and EC, is used as a filler to fill epoxy resin, the TC of blends can be much higher than that of blends containing more traditional fillers. However, the high EC of graphene limits its application in cases where electrical insulation is required. To address this challenge, a method for coating graphene sheets with an in situ grown Al2O3 layer has been proposed for the fabrication of epoxy-based composites with both high TC and low EC. In the presence of a cationic surfactant, a dense Al2O3 layer with a network structure can be formed on the surface of graphene sheets. When the total content of Al2O3 and graphene mixed filler reached 30 wt%, the TC of the epoxy composite reached 0.97 W m−1 K−1, while the EC remained above 1011 Ω·cm. Finite element simulations accurately predicted TC and EC values in accordance with experimental results. This material, with its combination of high TC and good insulation properties, exhibits excellent potential for microelectronic packaging applications.With the rapid development of the electronics industry, there is a growing demand for packaging materials that possess both high thermal conductivity (TC) and low electrical conductivity (EC). However, traditional insulating fillers such as boron nitride, aluminum nitride, and alumina (Al When graphene is used to fill epoxy resin, due to its high intrinsic EC, the low resistivity of the composites cannot meet the requirements for packaging materials [The high power density and frequency of modern electronic devices require effective heat dissipation in operation. This has stimulated the development of thermally conductive and electrically insulating materials. Traditional epoxy resin has low thermal conductivity (TC) and cannot meet the needs for practical application in thermal management . Filling2O3) ,5, graph2O3) ,7, and g2O3) ,9,10,11.aterials . TherefoThe surface treatment of graphene can be divided into minor molecule graft modification and surface deposition coating. The former requires grafting smaller molecular chains onto the ends or surface of graphene to reduce interface thermal resistance and improve compatibility between graphene and the matrix ,14,15,162) coating on graphene oxide (GO) was prepared [2 sandwich structure was 0.322 W m−1 K−1 at 1 wt% GO–SiO2 total loading, and the resistivity remained at 2.96 × 1011 Ω cm. In another example, tetraethyl orthosilicate (TEOS) was used as a precursor pre-grafted with 3-aminopropyl triethoxysilane (APTES) to generate nano-SiO2 in situ to coat graphene sheets and to enhance the compatibility between graphene and epoxy [−1 K−1 at 8 wt% loading. As a final example, Al2O3 deposited on a graphite sheet was used to prepare a thermally conductive and insulating phthalonitrile composite by hot pressing [−1 K−1 at a total loading of 20 wt%. In order to achieve both heat conduction and electrical insulation simultaneously, it is necessary to uniformly grow a coating layer with a specific structure on the surface of the fillers.Recently, the surface coating of graphene with different insulating layers has been achieved, effectively increasing the intrinsic resistivity of graphene. Using a sol-gel method, a silica  was purchased from Aladdin. Epoxy (E51), curing agent (593), and diluent (501) were purchased from Shanghai Lewei, China. Ethanol and distilled water were used on an instant basis.2O3@Graphene–CTAB. Another set of samples was prepared using the same method without the addition of CTAB, referred to as Al2O3@Graphene–NCTAB.An amount of 10 g of aluminum sulfate octadecahydrate was dissolved in 40 mL of deionized water, followed by the addition of 1 g of graphene sheets and 0.01 g of CTAB. The suspension was then mixed in a defoaming vacuum centrifuge at 1500 rpm for 5 min. In parallel, the stoichiometric ratio of NaOH was dissolved in 80 mL of deionized water. The NaOH solution was added dropwise into the mixture at a constant temperature of 60 °C with continuous stirring. The addition was divided into four parts, with an interval of 30 min between each addition, and the stirring was continued for 2 h after the complete addition. The resulting suspension was centrifuged and washed three times with deionized water and ethanol. Subsequently, the obtained sample was freeze-dried for 12 h. Finally, the freeze-dried alumina–graphene sample was ground, placed in a crucible, and heated at 540 °C for 3 h to obtain the desired filler, referred to as Al2O3@Graphene–CTAB was added and stirred in the defoaming vacuum mixer at 1500 rpm for 5 min. Finally, the mixture was poured into the poly tetra fluoroethylene (PTFE) mold and cured for 180 min at 120 °C to obtain composite samples.The epoxy resin, curing agent, and diluent were mixed with the calculated amount according to the ratio of 100:25:15. Then, the specified amount of AlA scanning electron microscope  was used to observe the morphology of the modified graphene and the fractured surface of the composites. Energy dispersive X-ray spectroscopy  was used to characterize the element distribution on the surface of the mixed fillers. The surface topography of the modified graphene was measured using transmission electron microscope in 100 kV . Thermogravimetric analysis  was used to measure the weight loss of the sample during the heating process in the air environment at 20 °C/min. The crystal form was measured by powder X-ray diffraction from 5–80° .λ = α × pC × ρ. λ, α, pC, and ρ represent TC, thermal diffusivity, specific heat capacity, and density, respectively. α was measured by a laser flash instrument  at room temperature on the sample of 12.7 mm in diameter. pC was obtained by differential scanning calorimetry  ranging from −5 °C to 50 °C at the heat rate of 5 °C/min. ρ was measured by an analytical balance  based on Archimedes’ principle. The electrical performance was characterized by a high resistance weak current tester .TC was calculated by the equation: 2O3 coating on the graphene surface morphology, SEM images were taken as shown in 2O3 coated graphene in the presence of CTAB surfactants. It was found that a dense network cladding structure was formed on the graphene surface. The cladding samples without CTAB in 2O3. With the slow addition of the alkaline solution, the newly generated aluminum hydroxide is more inclined to deposit on the graphene surface. In the absence of surfactants, aluminum hydroxide is more likely to grow on the existing crystal nuclei.In order to characterize the effect of the Al2O3 layer on the surface. This can be seen more clearly on the total elements distribution observed in 2O3 layer can be observed from EDX detection was used to verify the aluminum distribution on the graphene surface after the treatment in the presence of CTAB. As is shown in 2O3 is formed in situ. In order to have the exact proportions of different components, the TGA curve of the fillers was analyzed. As is shown in 2O3 started to lose weight at 549 °C and lost 24.56 wt% at 889 °C. This means that the graphene occupied about 25 wt% of the mixed filler. The higher the relative proportion of graphene in the mixed filler, the higher the TC of the powder, but also the higher the probability of forming an electrically conductive path inside the composites.Unlike with direct blending of different fillers, the respective filler content cannot be directly obtained in this way because Al2O3 coating. For instance, when preparing a composite with 15 wt% pure graphene using vacuum defoaming, the epoxy resin faced challenges in fully infiltrating the graphene structure. However, with the presence of the Al2O3 coating, even at a higher loading of 20 wt% Al2O3@graphene, the mixture exhibited excellent fluidity and uniform dispersion during the fabrication process. This improved moldability is attributed to the optimized interaction between the Al2O3 coating and the epoxy resin, enabling better wetting and penetration of the filler materials.Furthermore, the moldability of the composites was significantly enhanced with the incorporation of the Al2O3 powders obtained by precipitation sintering have the peaks of AlOOH and Al2O3 [2O3 sample was just used to make a comparison with the experimental group to characterize the change of peak position before and after coating. It is known from the previous TGA diagram that graphene will be thermally decomposed above 550 °C, so the heating temperature we chose was 540 °C. However, the aluminum hydroxide precipitated at this temperature cannot ultimately produce Al2O3, so the peak positions of AlOOH at 19°, 23°, and 66° can still be observed. The intrinsic peaks of graphene can be observed at 26.55° and 54.66° [2O3. Apparent differences in the morphology of the coating layer indicate that the impact is more on the generation site of aluminum hydroxide.XRD was used to further characterize the structure of the in situ coating layer as shown in nd Al2O3 . The decnd Al2O3 . Thus, td 54.66° , which p2O3@Graphene–CTAB, respectively. At lower magnification, it is evident that the graphene fillers were uniformly dispersed throughout the epoxy matrix, which can be attributed to the presence of the surface coating. At higher magnification, a dense Al2O3 layer can be observed on the surface of the graphene sheets. Notably, 2O3 layer epoxy matrix.2O3@Graphene-filled epoxy composite had a thermal weight loss of 58.58% and 15.35% at 484 °C and 617 °C, respectively, which corresponds to the thermal weight loss of pure epoxy. In addition, there was a 7.16% thermal weight loss at 831 °C, which corresponds to the partial weight loss of graphene. As is shown in 2O3-coated graphene started to lose thermal weight at 549 °C, indicating that there was also the decomposition of the filler before 617 °C. After heating to 1000 °C, the pure epoxy sample was almost completely burnt, while the 30 wt% Al2O3@Graphene-filled epoxy composite still had 18.76% residue. The quantity of the remaining white Al2O3 powder was consistent with the proportion of the input amount.In order to characterize the actual proportion of components in the composites, the TGA curves in air condition were also studied. The TGA curve of pure epoxy resin in 9 Ω·cm while maximizing the TC to ensure efficient heat dissipation. By increasing the graphene content in the composites, the TC increases while the volume resistivity gradually decreases. Therefore, it is crucial to carefully select the total filler content and coating ratio in order to achieve the desired properties.In the design of thermally conductive and electrically insulative packaging materials, achieving optimal thermal conductivity (TC) and electrical resistivity (EC) is of utmost importance. The insulation performance should meet a minimum value of 10The selection of an appropriate total filler content and coating ratio is critical in the design of composites for thermally conductive and electrically insulative packaging materials. Through careful optimization, it becomes possible to simultaneously achieve the desired TC and EC properties, ensuring that the materials meet the insulation performance requirements while maximizing thermal conductivity for efficient heat dissipation.−1 K−1, and the TC of the composite with 30 wt% filler reached 0.97 W m−1 K−1. Comparing the two sets of Al2O3@Graphene–CTAB and Al2O3@Graphene–NCTAB-filled epoxy composites, no significant difference of TC value can be noticed at low filler loading levels. Only at 30 wt% loading did the composite with CTAB show a significantly higher TC than that without CTAB. Al2O3 without surfactants in composites cannot form a net-like structure coating, as shown in 2O3 coating structure formed under the action of CTAB is very beneficial to maintain the dispersion of Al2O3@Graphene, as well as the insulative performance of composites.From 11 Ω·cm, which means that they can meet the requirements of insulation when applied. Below 20 wt% filler, the resistivity remained almost constant, indicating that the presence of the Al2O3 coating layer can well cover the electrically conductive graphene sheets. As the filler content increased further, even though the resistivity decreased rapidly, it was still two orders of magnitude higher than the critical value of 109 Ω·cm at the 30 wt% loading. 2O3@Graphene-filled epoxy composites. On the microscopic scale, a representative volume element (RVE) was constructed to describe the distribution of the fillers. The steady-state method was employed to establish a local microscopic heat and electricity transfer model and to homogenize and evaluate the effective TC and EC on the macro scale [In order to further understand the mechanisms of the thermal and electrical transfer, the finite element method (FEM) was used to simulate the TC and EC of Alro scale ,30,31.The finite element modeling used in this study is based on Ansys APDL. As a two-dimensional filler with high aspect ratio, graphene can be geometrically modeled with a rectangle. In a cubic RVE of a given size, the total number of two-dimensional rectangles is fixed. The relative size of the rectangle is adjusted to fit the composites with the specified filler content. It is equivalent to selecting a cube area of a specified volume according to requirements in a uniform system and studying this area separately to characterize the overall macroscopic TC and EC.2O3-coated graphene, three materials were defined as epoxy resin (matrix), graphene (filler), and Al2O3 (interphase). There was no mutual contact between the fillers. In order to know the influence of the Al2O3 coating on the TC and EC of the graphene-filled epoxy composites, an interphase with a specified mass fraction was added to the surface of the rectangle. According to the TGA data in 2O3@Graphene filler accounted for about 25%, so a 75% mass fraction interphase existed on the original surface of the two-dimensional disc. In order to describe the ternary composite system filled with Al6 Ω cm, it would result in an excessive difference in magnitude that prevents accurate calculations. Due to the high resistivity of the alumina coating on the graphene surface, the values for graphene were assigned according to the data in The thermal conductivity and electrical conductivity of graphene differ significantly from that of the matrix. Therefore, when assigning values in software, if the electrical conductivity of graphene is assigned as 102O3@Graphene–CTAB-filled epoxy composites. Heat transfer was studied in PLANE55. In order to obtain a random distribution model, x-y coordinates of the fillers and the angle formed by the filler length direction with the heat flow direction were used to describe the distribution state of the fillers. It can be seen that the position and orientation of fillers had a significant influence on the homogeneity of the temperature field. The location with fillers oriented in the heat transfer direction had a more protruding temperature field, meaning more rapid heat conduction due to the high TC of the fillers. The more random the filler distribution was, the more homogeneous the temperature field was.A steady-state heat transfer model was established by applying a temperature field to the left (high temperature of 323 °C) and right (low temperature of 298 °C) boundaries as shown in In order to describe the electrically conductive behavior of the filler inside the matrix, an electrical model was also established and the voltage field was simulated by Ansys APDL. The filler was randomly generated in the same way and the voltage field was calculated in PLANE230. For the same filler distribution model, the change of elements type can be used to calculate different physical fields. The steady-state method is also used to simulate the electrical field. Voltages of 5 V and 0 V were applied to the left and right sides of the model, and the average current density was calculated on the node to get the average resistivity of the whole.2O3 coating has simultaneously a higher TC than the matrix and a resistivity of up to 1013 Ω m, which makes the heat transfer and electrical conducting behavior different. It can be seen from 2O3 layer and then added into epoxy to prepare thermally conductive and electrically insulative composites. Under the control of the cationic surfactant CTAB and by mixing aluminum sulfate octadecahydrate, CTAB, and NaOH with graphene sheets following a certain procedure, a net-like form of Al2O3 was formed in situ and covered the surface of the graphene sheets, which changed the graphene from conductive to insulating. At 30 wt% mixed fillers, the TC of the epoxy composite reached 0.97 W m−1 K−1, while the electrical resistivity remained above 1011 Ω·cm. Compared with pure graphene filling, this dense mesh Al2O3 coating can ensure the electric insulation of graphene-filled epoxy composites and so meets special requirements for both high thermal conductivity and electrical insulation in practical application. A finite element simulation was performed by introducing an Al2O3 coating on the graphene. A very good agreement between the simulated and experimental TC was obtained, and the dependance of electrical resistivity on the filler loading was similar for the simulation and the tests. This work provides a guiding approach for the insulation of electrically conductive graphene and a new design method and possible industrial application of thermoset composites having both high TC and good insulation properties.In summary, graphene sheets were coated with an in situ formed Al


Mycobacterium abscessus (MAB) is a highly drug-resistant nontuberculous mycobacterium (NTM) that is an important cause of pulmonary disease (PD). Treatment is limited by the lack of active oral drugs and frequent adverse reactions. EBO is a novel oral, boron-containing antimicrobial that inhibits bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis. It has demonstrated encouraging in vitro and in vivo efficacy against MAB in small, limited preclinical studies and is currently in clinical development for the treatment of other NTM-PD. This study evaluated EBO minimal inhibitory concentrations (MIC) against a larger number of recent MAB isolates.MAB isolates collected in 2021 from the United States (n=122) from respiratory sources and in 2019-2022 from Europe (n=25) from respiratory and other sources were tested by broth microdilution according to Clinical and Laboratory Standards Institute guidelines using frozen microtiter panels manufactured by ThermoFisher against EBO and a panel of 13 antimicrobials with anti-MAB activity. MIC values were determined after 4-5 days of incubation; if an isolate was susceptible to clarithromycin (CLR) at day 5, it was reread at day 14 to assess for inducible macrolide resistance. Descriptive analyses were done on the MIC values.abscessus, 6 were bolletii, and 40 were massiliense. EBO MIC50/MIC90 for all isolates were 0.06/0.12 mg/L. MICs ranged from 0.03 - 0.25 mg/L and were consistent across subspecies (Table 1). MIC ranges, MIC50, and MIC90 values for all agents are summarized in Table 2. 95 isolates (64.6%) were CLR-resistant, consisting of 73 isolates with inducible and 22 with constitutive resistance. The EBO MIC50/MIC90 values for either CLR-resistant or amikacin (AMK)-resistant isolates remained 0.06/0.12 mg/L.Of the 147 MAB isolates, 101 were subspecies in vitro activity against MAB with MICs from 0.03-0.25 mg/L with similar activity against all subspecies. EBO MICs remained low against MAB isolates that were resistant to other agents, including CLR and AMK. These data demonstrate EBO’s promising in vitro activity versus MAB regardless of subspecies or resistance to current agents and support clinical evaluation of EBO as a therapeutic option for MAB disease.EBO demonstrated potent Tiffany Keepers White, PhD, AN2 Therapeutics: Employee|AN2 Therapeutics: Stocks/Bonds Charles L. Daley, MD, AN2: Advisor/Consultant|AN2: Grant/Research Support|Aztrazeneca: Advisor/Consultant|Beyond Air: Grant/Research Support|Bill and Melinda Gates Foundation: Data Monitoring Committee|Bugworks: Grant/Research Support|Genentech: Advisor/Consultant|Hyfe: Advisor/Consultant|Insmedd: Advisor/Consultant|Insmedd: Grant/Research Support|Juvabis: Grant/Research Support|Lilly: Data Monitoring Committee|MannKind: Advisor/Consultant|Matinas: Advisor/Consultant|Otsuka: Data Monitoring Committee|Paratek: Advisor/Consultant|Paratek: Grant/Research Support|Pfizer: Advisor/Consultant|Spero: Advisor/Consultant|Zambon: Advisor/Consultant