WWOX) participates in the generation of several cancer types, including lung cancer. However, whether these WWOX polymorphisms are related to the clinical risk of epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma is worthy of investigation. The present study examined the relationship between the WWOX single-nucleotide polymorphisms  and the clinicopathological factors in lung adenocarcinoma patients with or without EGFR mutations. We found that there was no significant difference in the genotype distribution of WWOX polymorphism between EGFR wild-type and EGFR mutant in patients with lung adenocarcinoma. Our results demonstrated that the presence of at least one G genotype (CG and GG) allele on WWOX rs3764340 was associated with a significantly higher risk of nearby lymph node involvement in those patients harboring EGFR mutations (odds ratio (OR) = 3.881, p = 0.010) compared with the CC genotype. Furthermore, in the subgroup of lung adenocarcinoma patients with the EGFR-L858R mutation, both WWOX rs3764340 C/G  and rs73569323 C/T polymorphisms  exhibited significant associations with the size of primary tumors and the invasion of adjacent tissues. In conclusion, these data indicate that WWOX SNPs may help predict tumor growth and invasion in patients with EGFR mutant lung adenocarcinoma, especially those with the EGFR-L858R mutant in Taiwan.Lung adenocarcinoma is the most common histological type of non-small cell lung cancer, which accounts for the majority of lung cancers. Previous studies have showed that dysregulation of WW domain-containing oxidoreductase ( Lung cancer has a higher incidence and mortality rate among cancers in the world . There aWWOX) gene is mapped to human chromosome 16q23.3–24.1, which spans approximately 1.1 megabases and consists of nine exons and eight introns [WWOX gene encompasses the FRA16D fragile region, which frequently undergoes chromosomal breaks and rearrangements in cancers [WWOX gene mainly encodes a protein of 414 amino acids (46 kDa). Its N-terminal region contains two tandem WW domains and its C-terminal region contains a catalytic domain homologous to short-chain dehydrogenase/reductase family proteins [WWOX protein has been shown to participate in a variety of cellular processes, such as DNA damage responses, cellular metabolism, and tumor suppression [WWOX exerts tumor suppressor effects through specific molecular actions that are mostly cell type specific [WWOX not only leads to tumorigenesis and cancer progression, but also causes genome instability and treatment difficulties [WWOX expression has been found to be associated with several cancers, such as breast cancers, thyroid cancer, oral cancer, lung cancer, and so on [The WW domain-containing oxidoreductase , are highly correlated with cancer susceptibility . Several cancers ,24,25,26WWOX genotyping, respectively. Lung adenocarcinoma is staged at the time of diagnosis according to the tumor/lymph node/metastasis (TNM) classification of malignant tumors of the American Joint Committee on Cancer. The size of the primary tumor and the invasion of adjacent tissues are described as T, T1-T4; the regional lymph nodes’ status is described as N; and distant metastasis is described as M [A total of 316 patients with lung adenocarcinoma were recruited at Chung Shan Medical University Hospital, Taiwan. All patient data, including gender, age and exposure to exogenous risk factors, such as smoking status, were obtained from medical records and questionnaires. Genomic DNA was collected from tumor specimens or whole blood specimens for EGFR gene sequencing or bed as M . The stuEGFR gene were amplified using the polymerase chain reaction (PCR) and were then subjected to the DNA sequencing reaction using the ABI PRISM 3130XL System  as described previously [Genomic DNA was extracted from the tumor tissues of patients with lung adenocarcinoma using the QIAamp Fast DNA Tissue Kit  according to the manufacturer’s instructions. Exons 18–21 of the eviously .WWOX polymorphisms  was examined using the TaqMan SNP genotyping assay and the ABI StepOnePlus Real-Time PCR system . For a negative control, each PCR reaction used distilled water instead of DNA in the reaction system.Genomic DNA was collected from whole-blood specimens of patients with lung adenocarcinoma using the QIAamp DNA Blood Mini Kit  according to the manufacturer’s protocols. The allelic identification of five p-value less than 0.05 was used to indicate statistical significance.The Mann–Whitney U test and Fisher’s exact test were used to compare the differences in clinical characteristics and genotype distribution frequencies between lung adenocarcinoma patients harboring the EGFR wild-type or mutation type in this study. Multiple logistic regression models after controlling for other variables were used to estimate the odds ratios (ORs) with corresponding 95% confidence intervals (CIs) of the association between the genotype frequencies and risk of EGFR types. All data were analyzed using SAS software, version 9.1 . A WWOX polymorphisms on lung adenocarcinoma risk, a total of 316 lung adenocarcinoma patients were enrolled. These lung adenocarcinoma patients were divided into patients with EGFR wild type  and patients with EGFR mutation  based on EGFR sequencing data. p < 0.001) and cigarette smoking status (p < 0.001) were observed between the patients with lung adenocarcinoma with the EGFR wild type and those with the EGFR mutation  and never smokers  in the EGFR mutant group . The genotype distributions and associations of WWOX gene between the lung adenocarcinoma patients with the EGFR wild type and those with the EGFR mutation were shown in WWOX polymorphisms between EGFR wild type and EGFR mutant in lung adenocarcinoma patients.Whole-blood specimens from the recruited 316 patients were collected and examined for the five WWOX gene in lung adenocarcinoma patients with two EGFR hotspot mutations, L858R (n = 89) and exon19 in-frame deletion (n = 91). In comparison with the EGFR wild type group, no significant association was found between WWOX polymorphisms and the two EGFR hotspot mutations in lung adenocarcinoma patients  allele on WWOX rs3764340 was associated with a significantly higher risk of nearby lymph node involvement in the patients harboring EGFR mutations  classification of the American Joint Committee on Cancer, an internationally recognized system . Next, wWWOX rs3764340 had the higher risk of nearby lymph nodes  of WWOX SNPs rs11545028, rs12918952, rs3764340, rs73569323, and rs383362 was not significantly different between wild-type and mutant EGFR lung adenocarcinoma patients. The existence of at least one G genotype (CG and GG) allele on WWOX rs3764340 exhibited a significant association with the involvement of nearby lymph nodes in the lung adenocarcinoma patients harboring EGFR mutations, particularly those with the EGFR L858R mutation. In the lung adenocarcinoma patients with the EGFR L858R mutation, the WWOX rs3764340 G genotype or rs73569323 T genotype of WWOX was shown to be associated with a significantly increase of primary tumor size and invasion of adjacent tissues. Our data suggest-that examining WWOX SNPs, rs3764340 and rs73569323, may help identify patients with lung adenocarcinoma with EGFR L858R mutations that have a higher risk of tumor size (T) and affected lymph node (N) status.In the present study, we found that the frequency of WWOX has been proven to perform tumor suppression through several molecular mechanisms in different cancers and diseases. The germline inactivation of the WWOX gene through homologous deletion can lead to human autosomal recessive diseases, such as the WWOX-related epileptic encephalopathy syndrome [WWOX expression in lung, breast, and bladder cancers are not only due to genomic alterations, such as loss of heterozygosity and homozygous deletions, but also epigenetic modifications, such as promoter hypermethylation. WWOX allele deletions have been found in 30.0% (three out of ten) lung adenocarcinomas [WWOX exon 6–8 deletion is reported to be high in Chinese NSCLC patients [WWOX gene and mutation of this gene may be related to NSCLC carcinogenesis [WWOX protein is lost or reduced in most NSCLCs and loss of WWOX expression is associated with to highly aggressive tumors using immunohistochemistry analysis. Multiple pathogenic copy number variations spanning of WWOX have been identified associated with neurological conditions [WWOX SNPs exhibited significant associations with the size of the primary tumor and the invasion of adjacent tissues in patients with EGFR mutant lung adenocarcinoma, especially those with EGFR-L858R mutant.syndrome . Iliopousyndrome  show tharcinomas . Indeed,ogenesis . Donati ogenesis  shows thnditions . Here, wWWOX polymorphisms showed no significant difference between wild type and mutant EGFR in lung adenocarcinoma patients. The FRA16D fragile region, which contains the WWOX gene, is a hot spot for genomic instability, prone to chromosomal breakage and copy number variations [WWOX gene and several missense polymorphisms in cancer cell lines and tumor tissues [WWOX SNPs, rs3764340 C/G and rs73569323 C/T, are highly associated with large tumor size and invasion of adjacent tissues in patients with EGFR-L858R mutant lung adenocarcinoma. However, we did not observe the association between the other WWOX SNPs and the risk of lung adenocarcinoma, which may be attributed to the limited samples.Lung adenocarcinoma has been shown to be related to many risk factors, such as genetic and environmental factors. Previous studies have shown that EGFR gene mutations occur more frequently in Asians than in Caucasians . Furtherriations ,10. The  tissues . Here weWWOX rs3764340 C/G polymorphism may be prone to the involvement of nearby lymph nodes, especially those with EGFR L858R mutation. In patients with EGFR-L858R mutant lung adenocarcinoma, WWOX rs3764340 C/G polymorphism is also associated with tumor growth and invasion. Previous studies have shown that the C/G genotype of WWOX rs3764340 is highly associated with differentiated thyroid carcinoma [WWOX to replace proline to alanine. We speculate that the variant allele 282 may affect the biological functions of WWOX, thereby making individuals susceptible to cancer.We found that the lung adenocarcinoma patients with both EGFR mutation and arcinoma , elevatearcinoma , and is WWOX rs73569323 polymorphism is positively associated with tumor size and invasion in patients with EGFR-L858R mutant lung adenocarcinoma. In contrast, WWOX rs73569323 SNP is found to decrease the risk of hepatocellular carcinoma [WWOX expression in clinical HNSCC tissues and miR-134 directly targets the 3′ UTR of WWOX gene in HNSCC cells [WWOX, whereas, anti-miR-134 increased WWOX expression in the small cell lung cancer cells. In addition, miR-24 also directly binds to the 3’ UTR of WWOX gene to inhibit gene expression in NSCLC cells [WWOX rs73569323 SNP may affect the various miRNA binding to 3′ UTR, leading to different expression and function of WWOX in different cancer types.Our data demonstrated that arcinoma . The rs7arcinoma . In headCC cells . InteresCC cells  show thaLC cells . TherefoWWOX SNPs and the clinicopathological characteristics of patients with lung adenocarcinoma. Since our study only analyzes the Taiwanese patient group, it may be necessary to conduct larger population-based studies in different ethnic groups for verification in the future. Moreover, it is necessary to study whether these polymorphic changes alter the function of WWOX protein in EGFR-mutated lung adenocarcinoma patients, especially L858R-mutated lung adenocarcinoma patients.There are some limitations in the present study. Further studies on larger cohorts are needed to confirm the association between these WWOX rs3764340 SNP is associated with a significantly increased risk of lymph node involvement in patients with lung adenocarcinoma carrying EGFR mutations. In addition, WWOX rs3764340 and rs73569323 polymorphisms are highly correlated with the size of the primary tumor and the invasion of adjacent tissues in Taiwanese lung adenocarcinoma patients carrying the EGFR-L858R mutation gene. Our data suggest that WWOX SNPs may help to predict tumor growth and invasion in patients with EGFR mutant lung adenocarcinoma, especially those with the EGFR-L858R mutant. In Taiwanese patients with EGFR-mutant lung adenocarcinoma, especially those with EGFR-L858R gene mutation, the underlying mechanism of the WWOX signaling cascade dysregulation is worthy of further exploration.The present results indicate that


Graft calcification after liver transplantation (LT) has seldom been reported, but almost of all previously reported cases have been attributed to graft dysfunction. We herein report two cases of graft calcification without liver dysfunction after living donor liver transplantation (LDLT). Two patients who underwent LDLT were found to have graft calcification in the early postoperative period (< 1 month). Calcification in the first case was found at the cut edge of the liver at post-operative day (POD) 10, showing a time-dependent increase in calcification severity. The second patient underwent hepatic artery re-anastomosis due to hepatic artery thrombosis on POD4 and received balloon-occluded retrograde transvenous obliteration of the splenic kidney shunt due to decreased portal vein blood flow on POD6. She was found to have diffuse hepatic calcification in the distant hepatic artery area at 1-month post-operation followed by gradual graft calcification at the resection margin at 6-month post-operation. Neither case showed post-operative graft dysfunction. Calcification of the liver graft after LDLT is likely rare, and graft calcification does not seem to affect the short-term liver function in LDLT cases. We recommend strictly controlling the warm/cold ischemia time and reducing the physical damage to the donor specimen as well as monitoring for early calcification by computed tomography. Liver transplantation (LT) is a treatment option for end-stage liver disease and acute liver failure. Various injuries, such as inflammation and primary benign or malignant tumor, have been reported to cause pathological liver calcification , 2. HepaWe herein report two cases with graft calcification without liver dysfunction after living donor liver transplantation (LDLT).A 50-year-old woman, diagnosed with Caroli’s disease, was performed underwent LDLT in 2009. She had a medical history of chronic renal failure without dialysis. Two years later, she underwent re-transplantation because of liver dysfunction caused by chronic anti-graft rejection. The living donor graft was transplanted with a blood-compatible extended left graft lobe. The graft weight/standard liver volume (GW/SLV) was 35.7%. The total operation time was 674 min, including 124 min of ischemia time. The blood loss was 5800 g. She had a normal liver function post re-transplantation.On POD10, computed tomography (CT) showed slight calcification in the transection area of the liver  Fig. a, b. On Graft calcification after LT is underreported with a rare rate. As of December 2020 in our department, we had performed 319 cases of LT, including 18 cases of deceased donor liver transplantation (DDLT). We experienced these two cases with graft calcification in early period (within 1 month) after LDLT without graft dysfunction. There might actually be more cases needed retrospective study.We reviewed the previously reported cases of graft calcification after LT Table . Tzimas Based on these previously reported findings, calcification after LT may be caused by ischemic injury. This may further affect the function of the graft and even the survival of the patients. Following IR, balanced apoptosis occurs, occasionally accompanied by necrosis of hepatocytes, which translates into cell swelling, distension of various cellular organelles, clumping and random degradation of nuclear DNA, extensive plasma membrane endocytosis and autophagy . In a prIschemic stress has been previously reported to induce calcium accumulation at the cellular level by an impaired energy metabolism and/or plasmalemmal alterations. The elevated intracellular calcium concentration causes cytoskeletal modification, which alters the cell shape, as well as activation of phospholipases, resulting in perpetuation of membrane damage and, finally, mitochondrial calcification .In addition, the cellular and molecular mechanisms underlying abnormal calcification following IR injury in LT have also been investigated , 9. AbunIn the present study, both cases underwent LDLT and were found to have graft calcification along the surgical cut margins without graft dysfunction. The calcification deposited in the edge of the incision wound seemed to be the result of hepatocyte apoptosis and necrosis caused by surgical manipulation. Furthermore, in case 2, the graft calcification was more obvious in the distant hepatic artery area than surgical cut margins and might have been caused by ischemic events due to HAT and a reduced portal vein blood flow after LDLT.As shown in Table Based on the short-term CT results (within 1 year), the severity and size of calcifications gradually increased with time. However, the long-term impact of such graft calcification on the transplanted liver remains unclear, so further long-term monitoring is necessary. The mechanism underlying the calcification occurring at the surgical incision surface is unclear, as most of the LDLT cases showed no calcification at the surgical incision area. Therefore, a retrospective investigation with LT cases should be performed in the future.In conclusion, calcification of the liver graft after LDLT is rare. While such graft calcification was not shown to affect the short-term liver function in LDLT cases, whether or not graft calcification affects the long-term liver function is a matter of concern. We recommend strictly controlling the warm/cold ischemia time and reducing the physical damage to the donor graft while performing early monitoring of the calcification by CT.


Several medicines, including cancer therapies, are known to alter the electrophysiological function of ventricular myocytes resulting in abnormal prolongation and dispersion of ventricular repolarization (quantified by multi-lead QTc measurement). This effect could be amplified by other concomitant factors . Usually, this condition results in higher risk of torsade de point and other life-threatening arrhythmias, related to unrecognized unpaired cardiac ventricular repolarization reserve (VRR). Being VRR a dynamic phenomenon, QT prolongation might often not be identified during the 10-s standard 12-lead ECG recording at rest, leaving the patient at increased risk for life-threatening event. We report the case of a 49-year woman, undergoing tamoxifen therapy for breast cancer, which alteration of ventricular repolarization reserve, persisting also after correction of concomitant recurrent hypokalemia, was evidenced only after manual measurements of the corrected QT (QTc) interval from selected intervals of the 12-lead ECG Holter monitoring. This otherwise missed finding was fundamental to drive the discontinuation of tamoxifen, shifting to another “safer” therapeutic option, and to avoid the use of potentially arrhythmogenic antibiotics when treating a bilateral pneumonia in recent COVID-19.The online version contains supplementary material available at 10.1007/s12012-021-09659-w. Drug-induced prolongation of ventricular repolarization is the most common and preventable cause of the so-called secondary Long QT syndrome (LQTS), which may imply high risk of life-threatening ventricular arrhythmic events –3. Otherhttps://ecis.jrc.ec.europa.eu/pdf/Breast_cancer_factsheet-Dec_2020.pdf] and it is the most frequently diagnosed cancer among US women too  plasma level, all QTc values were significantly  was statistically significant only during phases of sympathetic activation.In summary, retrospective systematic analysis of the four Holter recordings including comparative assessment during different rest and stress conditions, NREM and REM sleep (Table 1 in supplementary information), confirmed the following:At the end of December 2020, the patient was hospitalized for COVID-19 complicated by bilateral pneumonia Fig. a and diaBecause of the “a-priori” knowledge of impaired VR reserve, azithromycin and quinolones were avoided, and pneumonia was successfully treated with amoxicillin–clavulanic acid fixed dose combination. Hypokalemia was corrected with polarized KCl infusion until normalization.No significant QTc prolongation nor arrhythmia were observed during the hospitalization.Since its earliest definition, in 1998, the concept of “cardiac ventricular repolarization reserve” suggests that in the normal hearts there are complex homeostatic mechanisms to guarantee rapid and ordered VR and to avoid the risk of arrhythmogenic re-entrant circuits or triggered activity , 6. Such. Grades 3 and 4 are considered potential risk of life-threatening events [According to the National Cancer Institute (NCI) 4 degrees of QT prolongation induced by anticancer drugs must be considered, being: grade 1 (QTc 450–480 ms); grade 2 (QTc 481–500 ms); grade 3 (QTc > 501 ms on at least two ECG); grade 4 g events .torsade de point, by itself or when other conditions affecting the VRR  may overlap at same time [Among other cancer drugs, tamoxifen may induce QT interval prolongation and ame time , 10–12. ame time , 12. Altame time  because ame time –21, we fThe lack of normality ranges of circadian QTc values measured from Holter monitoring could be a limitation in interpreting our data. However, although peak values of QTc close to 500 ms (shortly after awakening), have been individually observed , the peaSince such abnormality was not evident at the standard 10-s 12-lead ECG recording, this, in our opinion, is a reasonable proof that in the reported case the evaluation of QTc variability provided by Holter monitoring was pivotal to discover an unknown and potentially arrhythmogenic reduction of VRR, likely enhanced by tamoxifen treatment, which for safety had to be withdrawn.Moreover, such observation was useful not only to guide the selection of a more appropriate cancer treatment, but also to prevent the contraindicated and potentially arrhythmogenic administration of azithromycin or quinolones when the patient needed additional treatment for bilateral pneumonia in COVID-19.As concerns the transient and asymptomatic ST and T-wave abnormalities observed during phases of sympathetic activation, their mechanistic interpretation is more difficult. An ischemic origin was reasonably ruled out by the absolute absence of angina symptomatology, their disappearance during maximum physical effort and by the asymmetric morphology of the negative T-waves. Being the patient asymptomatic, nuclear perfusion imaging scan was not performed, to avoid unnecessary additional irradiation. Theoretically it could be hypothesized that subclinical radiotherapy-induced myocardial fibrosis , sparselAlso, the cause of recurrent hypokalemia remains unclear, being renal and adrenal functions within normal range one could hypothesize a defect of gastrointestinal absorption, but that possibility has not been ascertained so far.The reported case confirms that before starting anticancer treatment, especially in female patients, cardiac ventricular repolarization reserve should be preliminarily assessed to prevent potentially arrhythmogenic drug-induced abnormal prolongation of the QTc interval and risk of life-threating arrhythmias. Whereas other methods can be somehow difficult to perform or ethically questionable , periodiLuckily in the reported case, although the QTc prolongation frequently exceeded 500 ms, major high-risk LQT syndromes were ruled out by the negative results of genetic tests and only rare single premature ventricular beats were observed. This suggests that, although impaired and generating transient ECG abnormality, the patient’s VRR was still sufficient to prevent the occurrence of dangerous ventricular arrhythmias and that probably a minor ion channel dysfunction has been unmasked only eventually by the overlapping of external factors such as tamoxifen-induced block of the IKr, enhanced by recurrent hypokalemia and only partially compensated by a gain of function of the IKs channel [Nevertheless, it was also evidenced that the recording of 10-s standard 12-lead ECG at rest , may be Therefore, we suggest that a baseline 12-lead ECG Holter monitoring, with accurate manual measurement of QTc, especially during sudden changes of HR under different day and night conditions, should be routinely performed at least before starting tamoxifen treatment [The cost–benefit ratio of follow-up Holter monitoring can change on the basis of local reimbursement policy. However, costs may be reduced with nowadays sensor and telematic transmission technologies and could favor the design of a randomized study as the next step, helpful to investigate the complexity of the physiological control of circadian modulation of ventricular repolarization rate dependence and to better define the normality range of QTc dynamics on larger populations .This is even more important nowadays, when the unpredictable occurrence of COVID-19 related complications may multiply the risk of arrhythmogenic events due to direct myocardial injury induced by the coronavirus and/or potential cardiotoxicity of drugs used to treat the cases with severe respiratory and cardiovascular complications, such as quinolone antibiotics, azithromycin, and antiretroviral therapies that may enhance the risk of adverse interactions and electrical instability increasing QT prolongation and transmural heterogeneity of ventricular repolarization. Such arrhythmogenic mechanisms can be enhanced by hypokalemia, cytokines-induced hyperactivation of cardiac sympathetic system, IL-6-induced inhibition of HERG potassium channels with consequent prolongation of ventricular action potentials and IL-6-induced inhibition of cytochrome P450 (CYP) 3A4 which may increase the bioavailability of the above mentioned QT-prolonging medications .Supplementary file1 (DOCX 354 kb)Below is the link to the electronic supplementary material.


Social insects use cuticular hydrocarbons for chemical recognition and communication. Cuticular hydrocarbons can also be exploited by parasites to their advantage for undermining host recognition systems. The small hive beetle (SHB) is a parasite of honey bee colonies but can also infest nests of other bee species. However, its chemical profile is still not known. For the first time, the present study investigated the SHB chemical profile and compared it with that of its honey bee host. The results show that the SHB has a low chemical profile that is similar to its honey bee host’s. However, while honey bees had a clear colony-specific chemical profile, SHBs did not. The generic chemical profile of the SHB is most likely linked to its free-flying behaviour in the field as these parasites are known to switch between host colonies, possibly limiting the acquisition of a colony specific chemical profile. Our findings also suggest that SHBs do not exploit any finely tuned chemical strategy to conceal their presence inside host colonies and probably rely on behavioural adaptations.Aethina tumida, which are parasites of honey bee, Apis mellifera, colonies, and other bee nests, is currently unknown. Here, adults of SHB and honey bee host workers were collected from the same field colonies and their CHC profiles were analysed using GC-MS. The chemical profiles of field-sampled SHBs were also compared with those of host-naive beetles reared in the laboratory. Laboratory-reared SHBs differed in their CHC profiles from field-sampled ones, which showed a more similar, but ten-fold lower, generic host CHC profile compared to host workers. While the data confirm colony-specific CHCs of honey bee workers, the profile of field-collected SHBs was not colony-specific. Adult SHBs often commute between different host colonies, thereby possibly preventing the acquisition of a colony-specific CHC profiles. An ester was exclusive to both groups of SHBs and might constitute an intraspecific recognition cue. Our data suggest that SHBs do not use any finely tuned chemical strategy to conceal their presence inside host colonies and instead probably rely on their hard exoskeleton and defence behaviours.Cuticular hydrocarbons (CHCs) cover insects’ bodies and play important roles in chemical communication, including nestmate recognition, for social insects. To enter colonies of a social host species, parasites may acquire host-specific CHCs or covertly maintain their own CHC profile by lowering its quantity. However, the chemical profile of small hive beetles (SHBs),  Discrimination between group members and foreign individuals represents a key feature of any social species. In social insect colonies, the ability to recognize colony members  is essential to maintain group integrity, avoid the exploitation of colony resources, and defend the colony from parasites and pathogens ,2,3. AltMaculinea rebeli which synthesises host-specific compounds before invading the nest of its ant host Myrmica schenki. Once inside the colony, it fine-tunes its chemical profile to the host’s colony odour by acquiring compounds, possibly through trophallaxis with the host [Many parasites have developed a number of strategies to deceive and exploit this recognition system, and to gain access to valuable colony resources ,12,13,14the host .Insects obtain most of their CHCs through synthesis that starts during the larval stage and slows down during the post-feeding stage; however, it will usually take several days after emergence to develop a complete CHC profile ,20,21. AAethina tumida, is a parasite of honey bee, Apis mellifera, colonies native to sub-Saharan Africa [The small hive beetle (SHB), n Africa . In 1996n Africa ,34,35. Wn Africa ; howevern Africa ,38, but n Africa . Adult Sn Africa . Within n Africa ,42, but n Africa . It has n Africa . HoweverHere, we characterized for the first time the chemical profile of the adult SHB and investigated the similarities with its honey bee host profile. Based on previous observations of lower aggressiveness exhibited by honey bees towards nestmate SHBs , and theA. mellifera, colonies in three different apiaries around Auburn, AL, USA in Summer 2019. Honey bee workers were collected from brood frames and SHB from entire hives of local naturally honey bee colonies using aspirators [Adult SHBs  and adult honey bee workers  were collected from five different queenright honey bee, pirators . ExperimTo obtain laboratory-reared bee naive individuals, SHB adults were collected from naturally infested honey bee colonies and used to initiate laboratory rearing following standard protocols . In brieCHC extracts were obtained by washing each honey bee worker in 1 mL of hexane and each SHB in 0.5 mL of hexane for 15 min. The different amounts of solvents were reflecting differences in body size. Then, the extracts were allowed to evaporate and dried samples were covered with foil and transported to Italy for coupled gas–chromatography mass spectrometry analysis (GS-MS).Dried extracts of all specimens  were re-suspended in 100 μL of pentane and transferred to a conical glass insert inside the original vial used for extraction. The solvent was then dried under a stream of nitrogen and the SHB samples were re-suspended in 20 μL of heptane with 70 ng/μL of heptadecane (n-C17) as the internal standard. For the honey bee worker samples, 80 μL of heptane with 70 ng/μL of heptadecane (n-C17) as internal standard. The final volume of resuspension was quadrupled for honey bee samples since preliminary analyses of a few specimens showed peak saturation for honey bee extracts re-suspended in 20 μL of heptane added with heptadecane due to the high quantity of CHCs extracted from honey bee workers. One μL of the extract was injected in a Hewlett Packard  5890A gas chromatograph (GC) coupled to an HP 5970 mass selective detector (using 70 eV electronic ionization source). A fused ZB-WAX-PLUS (Zebron) silica capillary column (60 m × 0.25 mm × 0.25 mm) was installed in the GC. The injector port and transfer line temperatures were set at 200 °C and the carrier gas was helium (at 20 PSI head pressure). The temperature protocol was from 50 °C to 320 °C at a rate of 10 °C/min, and the final temperature was kept for 5 min. Injections were performed in splitless mode . Data acquisition and analysis were performed using the Chem Station G1701 BA (version B.01.00)—Copyright© Hewlett-Packard 1989–1998. Compounds corresponding to different peaks in each chromatogram were identified on the basis of their retention time and mass spectra. Mass spectra were compared with mass spectral electronic libraries .The amount of each compound was evaluated by dividing its abundance by the abundance of n-C17 (multiplied by 4 for honey bee workers because of the higher dilution). The resulting amount was transformed by the method provided by Aitchison , which ap values from multiple Kruskal–Wallis tests were adjusted by using the Benjamini and Hochberg procedure implemented in the p.adjust function of the stat R package. To detect if SHB changed its profile after entering a honey bee colony to match that of the host chemical, we calculated chemical dissimilarity among the signature centroid of honey bee workers (average transformed amount of each compound), host-naive SHBs reared in the laboratory, and SHBs collected from host colonies in the field. Chemical dissimilarity was calculated using the Bray–Curtis dissimilarity (vegdist function of the vegan R package). Dissimilarities to honey bee worker centroid to laboratory-reared and field-collected SHBs were compared with a Mann–Whitney test.Univariate and multivariate analyses were applied to compare the possibility of attributing honey bees and SHBs to the colony they belong to based on chemical composition. With this aim, first was performed a Partial Least Square Discriminant Analysis (PLSDA) as implemented in the mixOmics R package . As a grThe possibility to attribute honey bee workers to their maternal colonies based on the typical colony profiles was tested by a jackknife procedure where a sparse PLSDA (SPLSDA), more conservative than a PLSDA since it allows the inclusion of a reduced number of variables per each discriminant component (ten variables in our assessment), was performed on all the specimens but one using colony membership as a grouping variable. Then, colony membership of the excluded specimen was predicted on the basis of their CHCs composition. Colony membership of field-collected SHBs was predicted on the full SPLSDA model obtained for honey bee workers. The percentage of correctly attributed cases was used as a measure of the possibility to blindly attribute individuals to their colonies. Finally, the overall quantity of compounds (not transformed by Aitchison formula) was compared among laboratory-reared SHBs, field-collected SHBs, and honey bee workers by using a Kruskal–Wallis test with post hoc comparisons. All calculations were performed using the program R .Laboratory-reared and field-collected SHBs had a low chemical profile that was similar to its honey bee host . LaboratA Partial Least Square Discriminant Analysis (PLSDA) separating 11 groups of honey bees and SHBs  based on their CHC signatures showed that the laboratory-reared SHBs, field-collected SHBs, and honey bees formed three distinct clusters . Most ofField-collected SHBs showed changes in chemical composition compared to laboratory-reared ones, thereby resembling a more generic honey bee profile. Indeed, a jackknife procedure performed to blindly attribute each honey bee sample to a colony based on comparing each chemical profile with a SPLSDA model constructed on all other honey bees showed that 68.1% of honey bees were correctly classified. Conversely, when profiles of SHBs were attributed to a colony based on SPLSDA models constructed on honey bee profiles, only 14.6% of individuals were correctly classified. Accordingly, a the PLSDA where honey bees and field-collected SHBs were grouped to their colony membership showed that honey bee workers form distinct groups based on the two first discriminant components alone ; conversp < 0.001, Chemical dissimilarity, calculated as the pairwise Bray–Curtis dissimilarity from the chemical signature centroid of field-collected honey bee workers, was significantly higher for the CHC profiles of laboratory-reared SHBs than for field-collected SHBs . Post hoc comparisons showed a significant effect in all pairwise comparisons .The overall amount of chemicals significantly differed between laboratory-reared SHBs, field-collected SHBs, and honey bee workers  was exclusively found in both laboratory-reared and field-collected SHBs, but not in honey bee workers. This ester may, therefore, constitute an intraspecific SHB recognition cue, whose actual role for communication appears worthy of investigation.The present work provides the first characterization of adult SHB chemical profiles in comparison to honey bee host workers. Our data showed that adult SHBs possess a generic honey bee host CHC profile. In the field, the SHB CHC profile was not host colony-specific, probably due to adult beetles commuting between host colonies. This suggests that SHBs do not use a finely tuned chemical mimicry to conceal their presence inside a honey bee colony. The ten-fold lower CHC profiles of field-collected adult SHB compared to honey bee workers might, nevertheless, constitute an adaptation to at least partly evade nestmate recognition.


Although current cutting-edge techniques enable very early detection of tumors, profiling whether a tumor has already begun to spread and where it has attempted to colonize remains a major hurdle. Indeed, metastatic seeding events exhibit remarkable temporal and spatial heterogeneity, wherein the origin (primary site) and destination (metastatic site) are highly dynamic. For example, liver metastasis is particularly common and remains a leading cause of mortality3. Primary cancers are diverse and can include gastrointestinal cancer, such as colorectal cancer and pancreatic cancer, as well as cancers of extraperitoneal origin, such as breast cancer, which has heterogeneous clinical phenotypes and a range of therapeutic responses. Because existing knowledge of cross-tissue metastasis remains far from complete, the generation of a unified pan-cancer metastasis map remains a pressing need. In this perspective, we outline the growing advances in metastasis biology, with a focus on the challenges in using high-throughput technologies and the state-of-the-art theories among the metastasis research community.Cancer metastasis remains one of the most confounding questions in oncology4. This process requires several key steps involving cancer cells leaving the tumor, entering the vessels, surviving the systemic immune system, and finally colonizing distal organs5. These key aspects of cancer seeding are regarded as the hallmarks of metastasis6. In detail, at the initial stage of metastasis, cancer cells often have high motility and invasiveness, thus enabling later intravascular dissemination and transport7. Interestingly, the balance between circulating cancer cells and the systemic immune system can determine the fate of metastatic cells8. Later, at the colonization stage, cancer cells show complex interactions with the microenvironment6. For example, hepatic metastatic cells engage in cross-talk with a broad spectrum of liver resident cells , which have opposing roles in the progression of metastasis9. Recent evidence has also highlighted that the primary tumor provides a microenvironment that can precondition metastatic cancer cells, thus favoring their growth and disrupting the fragile balance10. The most recent high-throughput omics technologies can now be leveraged to profile metastatic cell landscapes across different conditions and cancer types, thus yielding data-driven insights to refine understanding of the nature of metastasis.Metastasis has long been appreciated to be the most lethal complication of cancer progression16. For instance, hepatic metastatic cancer cells inherit multiple genetic subclones from primary colorectal cancer cells11. Moreover, ~65% of metastases have been reported to originate from independent subclones, thus highlighting the complicated seeding patterns of colorectal cancer cells17. The route of colorectal hepatic metastasis has been classified into 3 models: initiation from primary tumors (~62.3%), lymph node metastases (~36.1%), and intrahepatic metastases (~1.6%)18. However, almost all these findings were based on a “gene-centric” model, which does not consider the effects of epigenetic cis-regulatory regions. Further exploration of whether the alterations occur at enhancers or transcription factor binding sites should provide a unique integrated perspective on the complicated linkage between the epigenome and aggressive phenotypes.In the context of metastasis biology, an important question naturally arises: how do metastatic cancer cells evolve after arriving at a new location? Paradoxically, genomic profiling has indicated that primary and metastatic cells generally have similar profiles16. At the epigenetic level, hepatic metastatic cancer cells shift their gene transcription programs and show reprogramming of the landscape of enhancer binding by liver-specific transcription factors19. Another independent group has also reported that metastatic cells undergo chromatin remodeling in the liver20, thereby highlighting the specific transcriptional states shaped by the liver microenvironment.Beyond genetic diversity, another confounding question regarding metastasis is the evolution of cancer cell state. Colonized cancer cells are likely to be “unfamiliar” with the new environment and may potentially initiate an adaptation program. For example, in the liver, the central organ of glycolytic and drug metabolism, metastatic cancer cells are exposed to toxins and nutrients. Consequently, these metastatic tumors exhibit strong activation of drug metabolism and PPAR signaling pathwaysThese recently generated data have raised more hypotheses than they have answered. Many microenvironmental factors  appear to associate with cancer cell phenotypes, but little is known regarding whether those changes are passengers or drivers. The overall transcriptional state is reprogrammed, but whether this state is dominated by the majority or minority of cancer cell subpopulations remains an interesting question to be answered. The resolution of these fundamental questions should open exciting new paths to in-depth understanding of metastatic cell states and regulation, an area of remarkable importance in both metastasis biology and anti-metastasis therapy.7. Mouse model evidence has indicated that liver metastasis creates an “immune desert” environment with significantly diminished T cell diversity and function21. That work has explained why immunotherapy appears to fail when metastasis occurs and has raised the possibility of combining radiotherapy with immunotherapy to promote antitumor immunity21. We and Li’s laboratories have recently reported the spatiotemporal immune landscape of colorectal cancer liver metastasis23. We observed that the hepatic metastatic microenvironment is enriched in MRC1+ CCL18+ M2-like macrophages. Unexpectedly, these macrophage subsets show a broad range of activated metabolism related pathways. To address the challenges of investigating single-cell metabolism, we have developed the pipeline scMetabolism  and used it to confirm the sharp increase in metabolic activity, such as phenylalanine metabolism. These patient-derived observations are consistent with our mouse model data22, thus indicating that metastasis-specific immune cell subsets and immunometabolism might potentially be conserved across species.Along with (epi)genomic, transcriptomic, and proteomic dysregulation, microenvironmental alterations have emerged as a hallmark of metastasis24. In the context of liver metastasis, several immune components have been demonstrated to be essential for the PMN, such as neutrophils25 and macrophages26. However, almost all evidence has originated from laboratory conditions. Higher-level evidence from clinical samples remains needed, although acquiring such samples and defining the PMN state remains challenging. Innovations such as high-resolution imaging  and liquid biopsy (detection of circulating biomarkers predictive of early metastasis) are expected to markedly improve the possibility of profiling the PMN and thus deepen understanding of their functional outcomes.A crucial step in cancer seeding is the establishment of a pre-metastatic niche (PMN) whose formation can be divided into 4 distinct steps: priming, licensing, initiation, and progression28. The importance of combinational therapy  or neoadjuvant therapy  against metastases is increasingly being recognized. In contrast, independent studies have shown that neoadjuvant therapy may also initiate the metastasis cascade by driving the formation of an unfavorable metastatic microenvironment30 and inducing the generation of resistant cancer stem cells31. These seemingly paradoxical observations highlight the complexity of metastatic cancer cell fitness with respect to their environmental conditions and prompt the fundamental question of how to trace metastatic seeding under distinct pharmacological conditions.Although therapeutic strategies against metastasis are highly diverse across tumors, the consensus is that most metastases significantly decrease the efficacy of existing treatments23. Our results have shown that neoadjuvant chemotherapy restores the balance of anti-tumor immunity by depleting MRC1+ CCL18+ macrophages and increasing cytotoxic CD8+ T cells in responsive patients. In contrast, progressive disease and stable disease samples show higher proportion of suppressive immune cells. This result is generally consistent with Li’s findings23. These data collectively emphasize the importance of infiltrated immune cells to the sensitivity of neoadjuvant chemotherapy and suggest potential opportunities to explore the mechanisms of metastasis-immune interaction via single-cell profiling and computational modeling. Therefore, to improve knowledge of why chemotherapy selectively benefits certain subsets of patients, understanding of not only the cancer cells themselves but also their interaction dynamics across time and space is necessary.To overcome this challenge, our group and Li’s group have recently used scRNA-seq to compare the immune microenvironment of neoadjuvant chemotherapy-treated and treatment-naïve colorectal cancer liver metastasis samplesHowever, knowledge regarding the mechanisms underlying metastatic drug responsive processes remains scarce. Although scRNA-seq can capture the transcriptomes and cell states of both immune cells and cancer cells, an essential question remains: do the immune cells and cancer cells co-evolve during therapy? Moreover, how does neoadjuvant chemotherapy reprogram the state of cancer cells? The local ecosystem of metastatic tumors can vary dramatically during long-term exposure to therapeutic stimulation, wherein alterations in the blood supply, cancer cell and immune metabolism, and intercellular cross-talk are all likely to reshape the therapeutic responses. Thus, longitudinal follow up and profiling of the metastatic ecosystem will be necessary in the future.Figure 1A). Although several efforts have attempted to decode the genetic evolution during metastasis across common cancer types32, the major goal of decoding the route of metastasis remains far from being met. For example, few studies have focused on explaining why metastases in the same organ can originate from distinct primary cancers. Similarly, why the same primary tumors can colonize different distal organs and how those metastases are specifically transcriptionally reprogrammed are poorly understood. The necessary pan-cancer sampling strategy is practically challenging, because it requires long-term collection of non-invasive biopsy samples; however, it may make finally assembling an atlas possible.First, the longitudinal collection of clinical metastatic samples and their matched primary tumors is particularly informative for identifying the evolutionary patterns of metastasis  across multiple timescales . Future technological advances are likely to improve the throughput, dimension, and resolution. Therefore, bioinformatics algorithms must urgently be updated to analyze such large data. We believe that future 34. However, those methods largely rely on the construction of gene-edited cell line models. With patient-derived xenografts and organoids, which can closely reflect the heterogeneity of clinical samples, the genotypes and phenotypes of human metastatic cancer cells can now be charted. We believe that the surge in of advanced high-throughput sequencing technologies and lineage tracing systems will advance metastasis biology beyond the current paradigms.Third, a more advanced lineage tracing tool remains needed to track metastatic seeding. The current Cas9-based single-cell tracing system enables monitoring of metastatic cells with months of growth and disseminationFigure 1B). The future database or resource may potentially enable the following: (1) systemic trajectory tracing of primary and metastatic cancers; (2) unified analysis of multi-omics primary-metastasis data; and (3) drug response prediction based on the multi-omics profile. We believe that these efforts will be particularly valuable for the analysis of multi-omics big data and new hypothesis generation.Finally, the development of a pan-cancer metastasis resource will support a broad spectrum of metastasis research and enhance the rapid discovery of novel metastasis biology (Metastasis research is rapidly advancing. The surging advances in single-cell spatial omics, computational biology, and data science are providing unprecedented opportunities for data generation, analysis, and theoretical development. Progress in exploring the molecular principles controlling cancer seeding will advance the field from viewing a fixed snapshot to a dynamic perspective on metastatic evolution. Simultaneously, the generation of unbiased, large-scale, high-throughput data is poised to facilitate the generation of a map of pan-cancer metastasis and is expected to drive an exciting research paradigm shift in the coming years.


Branchial cleft cysts are congenital anomalies which develop in utero, most commonly arising from the second branchial cleft. They are often asymptomatic lateral neck masses but can enlarge and become symptomatic in the setting of infection. The cystic cavity can form a potential space which can harbor infection and, in rare cases, malignant spread of primary tumors. Herein, we present a rare case of a 28-year-old male with an enlarging branchial cleft cyst of six months duration following an upper respiratory infection. Routine post-surgical histopathological examination of the excised mass revealed metastatic papillary thyroid carcinoma. To our knowledge, this case is one of five cases reported within the primary literature. This case draws attention to the occult nature of papillary thyroid carcinoma and the importance of routine histopathological examination of seemingly benign surgically excised lesions. Branchial cleft cysts are the most common congenital neck masses arising laterally . ApproxiA 28-year-old male was referred to the otolaryngology clinic for a mildly tender lateral neck mass that first appeared six months prior to presentation. The mass steadily grew in size. It was initially accompanied by mild sinus congestion without constitutional symptoms, dysphagia, dysphonia, shortness of breath, or any other unusual symptoms. Aside from a body mass index of 49, the patient had no pertinent medical history. He reported a 10-pack-year smoking history and social alcohol consumption limited to three to four beers weekly. His family history was significant for unspecified lung malignancy. Physical examination revealed a 5-cm cervical mass inferior to the mandible on the lateral neck. There was no stridor, the trachea was midline, and the thyroid was non-tender without palpable nodules. No other masses were appreciated on palpation of the neck. Contrast-enhanced computed tomography (CT) of the head and neck revealed a 6.5 cm x 4 cm cystic mass at level 3, consistent with a second branchial cleft cyst Figures , 1B.Fine needle aspiration of the cystic content was benign and without malignant cells. The branchial cleft cyst was surgically excised under general anesthesia without complications and the excised specimen was forwarded to pathology for further evaluation. Histopathological examination revealed a cystic component, delineated by squamous and ciliated respiratory epithelium, containing papillary structures with loose fibrovascular cores and hyperchromatic nuclei consistent with classic PTC Figures -2D.Immunohistochemical staining was cytokeratin 7 (CK 7) positive and thyroid transcription factor-1 (TTF-1) positive, commensurate with a diagnosis of metastatic PTC. Subsequent ultrasound of the thyroid revealed a 1.5-cm solid isoechoic nodule in the right lobe with characteristics correlating with a thyroid imaging reporting and data system (TI-RADS) 5 lesion. Fine need aspiration of the contents revealed thyroid follicle cells showing features suspicious for papillary carcinoma.The patient underwent a total thyroidectomy and was noted to have three separate foci of well-differentiated PTC in the right lobe with a small micro-carcinoma focus within the left lobe. Enlarged lymphadenopathy discovered in level 6 of the neck was identified as a site of metastases with extension outside of the lymph node into the soft tissue. Other foci were contained within the surgical margins. Following thyroidectomy, the patient was treated by a multi-disciplinary care team consisting of representatives from otolaryngology, endocrinology, and radiation oncology. At the patient’s six-week follow-up appointment, the patient had no particular neck complaints. The patient elected for aggressive radioactive iodine ablation and long-term thyroid suppression.Branchial cleft cysts are embryological remnants that fail to close in utero ,3. ThesePTCs are common thyroid tumors with metastatic potential. These tumors share an occult clinical picture consistent with asymptomatic congenital neck masses . Many inPTC metastasis within the confines of a branchial cleft cyst has been reported in the literature, albeit infrequently -14. A reFortunately, in this case, the patient sought care when the branchial cleft cyst became symptomatic following an upper respiratory illness. In the absence of symptoms, the patient would have been unlikely to seek medical treatment. This patient had limited financial resources and time away from work which hindered his ability to seek routine medical care. Further, the patient was morbidly obese with a body mass index of 49, thereby obfuscating thyroid testing and limiting detection even with routine thyroid physical examination . Taken tMetastatic dissemination of PTC within the confines of a second branchial cleft cyst is a rare finding with only four other cases previously documented in the literature. This case report serves to illustrate the importance of routine pathological examination of surgical specimens, even in those lesions of seemingly benign etiology. While PTC is amenable to treatment if identified early, harboring of a metastatic lesion within a benign congenital mass may lead to delayed treatment and reduced overall survival. More research is needed to discern the impact of socioeconomic status in concert with this unique clinical scenario.


Skeletal muscle is considered an essential tissue involved in many physiological functions such as metabolism, thermoregulation, respiration, and locomotion. It has become clear that genetic and environmental factors are intricately involved in regulating skeletal muscle volume or myofiber size. The complexity of the orchestration of the signaling pathways that regulate these factors is intriguing. Muscle biologists strive to elucidate the myriad of signaling interactions critical for muscle volume and homeostasis. In this topic, we aimed to gain new insights into the basis of muscle volumetric and homeostatic regulation. We collected nine papers that embody novel or important concepts to skeletal myology. The three main areas compose the manuscripts: adult myogenesis, skeletal muscle mass regulation, and cancer-induced muscle weakness.Cirillo et al. The authors have previously reported that hypoxia-preconditioning upregulated both HIF-1α and Wnt7a followed by enhanced hypertrophy  in myogenesis by Tan et al. demonstrates that the knockdown of RNA-dependent protein kinase-like ER eukaryotic translation initiation factor 2 alpha kinase (PERK), one of the three UPR signaling branches, exaggerated myotube formation through perturbing micro RNA (miRNA) networks in the immortalized mouse skeletal myoblast cell line C2C12 culture. While in vivo confirmation is required, the results propose the unique relationship between UPR and miRNA in the context of myogenic differentiation.The unfolded protein response (UPR), an evolutionarily conserved intracellular signaling mechanism, is an important event involved in adult myogenesis  isoforms expression. Thus, the results suggest that Mdfi plays a positive role in myogenic differentiation under a culture setting.Myod family inhibitor (Mdfi or I-mfa) is a myogenic repressor  affects anabolism and catabolism processes including mechanistic target of rapamycin (mTOR) signaling. Considerable effort has focused on the role of branched-chain amino acids (BCAAs) trafficking in coordinating protein turnover.This Research Topic includes two comprehensive review articles discussing skeletal muscle homeostasis. van Ingen and Kirby summarized the current concept and topic of nuclear mechanobiology related to skeletal muscle homeostasis and function.The second review article discusses transduction of mechanical signals to the nucleus. This research area has recently received attention in skeletal myology. A mechanical network in the nucleoskeleton and cytoskeleton is a crucial element to understand cellular mechanobiology. Proper mechanotransduction to the nucleus can initiate appropriate response and adaptation of muscle cells to local environmental changes. Straughn et al. demonstrated that the anti-tumorigenic compound Withaferin A (WFA), purified from the plant Withania somnifera, directly acts to the skeletal muscle to induce hypertrophic adaptation under ovarian cancer environment. Thus, WFA could be a potential therapeutic means to ameliorate both ovarian cancer and cancer-related muscle wasting.Cancer biology is now an important field of skeletal myology because cancer development causes substantial volumetric reduction of skeletal muscle in patients. There are three articles in this collection that examine the crosstalk of skeletal muscle with cancer. Halle et al. demonstrated that passive stretch in C2C12 myotubes could prevent diminished protein synthesis signaling and fast-type myosin expression induced by C26 tumor-derived factors in a cultured setting. This study provides initial evidence that mechanical stimulus could counteract cancer-induced muscle deconditioning.Colon-26 (C26) tumor cells-derived factor inhibits myotube growth signals. Since passive stretch has been identified to increase protein synthesis and muscle volume, Mallard et al. reviewed the research to understand the alterations in skeletal muscle in breast cancer patients, particularly as a consequence of chemotherapy, and then prospectively described potential mechanisms based on multiple types of cancer-related conditions. This review article also includes information regarding the fundamental signaling pathways that regulate skeletal muscle volume.Finally, We hope that our Research Topic is informative to advance the field of skeletal muscle research. Lastly, we thank the authors for their contributions to this collection.YO wrote the first draft of the manuscript. YO, SS, YSG, and STA wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.SS was supported by Louisiana Board of Regents Support Fund (LEQSF (2017-20)-RD-A-22).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Electrical impedance tomography (EIT) is noninvasive and can be used at the bedside for real-time evaluation to identify ventilation distribution of infected lungs. This review briefly describes the basic principle of EIT and summarizes the latest findings on its potential contribution to lung protective strategies in coronavirus disease 2019 patients. Additionally, experimental approaches for detecting the distribution of pulmonary blood flow in coronavirus disease 2019 patients are presented. The findings underscore the role of EIT in determining lung protective strategies for coronavirus disease 2019-associated acute respiratory distress syndrome. Acute respiratory distress syndrome (ARDS) is characterized by life-threatening hypoxemia caused by hyperpermeability of pulmonary capillaries resulting from various background diseases or conditions. Although mechanical ventilation is required for ARDS diagnosis and treatment, it has become obvious that ventilator-associated lung injury plays an important role in the development of multiple organ failure, which is the cause of death for ARDS. Large-scale clinical trials have demonstrated that low tidal volume ventilation (LTV), which limits both tidal volume and plateau pressure, improves ARDS prognosis. Currently, LTV is one of lung protection strategies and is essential for treating ARDS.(1). In an animal model, increased dynamic strain was more associated with the development of pulmonary edema, derangement of lung mechanics, and higher mortality than static strain (2). However, monitoring measures to assess regional ventilation homogeneity in critically ill patients at bedside are limited. Computed tomography is a standard test for examining ventilation and aeration of the lungs, but transportation of critically ill patients, radiation exposure, and limitations to static evaluation are the major obstacles for its frequent use in the clinical practice. Developing a new testing method that can be performed repeatedly at bedside is warranted.When gas exchange cannot be maintained by LTV, early implementation of prone positioning acts as rescue therapy and improves the prognosis. It is well known that prone positioning recruits collapsed regions and improves oxygenation. Recent studies have suggested that prone positioning promotes homogeneous ventilation distribution and might have a lung protective effect. If the lungs are evenly ventilated by reducing regional overdistention and recruiting the collapsed region, local stress and strain can be ameliorated, resulting in the prevention of ventilator-induced lung injury. The importance of homogeneity of regional ventilation has been increasingly recognized. It has been reported that lung inhomogeneity during tidal ventilation increases dynamic lung strain (3). Briefly, the impedance value is measured by applying a small alternating current through electrodes implemented in the EIT belt, which is normally placed at the fifth to sixth intercostal level of the patient’s chest  measures and visualizes the impedance changes generated by ventilation in real time. The principle of measurement is described elsewhere ’s chest . SequentEIT is radiation free, and a dynamic regional ventilation profile can be monitored at the bedside every time the ventilator settings are changed.2O). The whole procedure was monitored and recorded using EIT, and the saved impedance data were analyzed using software .EIT provides reliable information to detect alveolar overdistention and collapse. To assess the homogeneity of ventilation distribution, the patient was paralyzed, and incremental and decremental PEEP trials were performed at a fixed driving pressure (10-12 cmH(4), providing useful information when titrating personalized PEEP (5). EIT might help evaluate the possibility of lung recruitment maneuvers and estimate recruitable alveolar collapse (6). Prone positioning is a rescue therapy for patients who do not respond to lung protective ventilation. We performed prone positioning for a patient with ARDS who could not maintain gas exchange with LTV and observed the process of the change in regional ventilation distribution using EIT (7). Initially, ventilation was shifted to the mid-ventral region, and the dorsal area completely collapsed. Twenty minutes after the initiation of prone positioning, ventilation distribution became more homogeneous, and oxygenation improved dramatically. The homogeneity of ventilation was stabilized after the second session, and the patient survived. Prone positioning basically provides homogeneous ventilation distribution in ARDS lungs, but temporary overdistention in the dorsal region has also been observed immediately after initiation (8). The response to prone positioning varies among cases and the close monitoring using EIT provides information to improve its efficacy.In severe ARDS patients, EIT is used to optimize mechanical ventilation settings The outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emergent threat to the current global health. In Japan approximately 1.6% of patients with coronavirus disease 2019 (COVID-19) are severe, defined by the requirement of tracheal intubation and mechanical ventilation. It has been reported that COVID-19 pneumonia can cause ARDS. Therefore, it is necessary for COVID-19-associated ARDS to implement the same lung protection strategy described earlier. However, a recent study paid special attention to lung protection strategies for COVID-19. EIT plays a role in determining lung protective strategies for COVID-19-associated ARDS.(9). Conceptually, type L is characterized by low elastance, low ventilation-perfusion ratio, and low lung weight and recruitability, whereas type H is defined as high elastance, high right to left shunt, and high lung weight and recruitability. Recruitability is defined as the possibility to open up collapsed areas of the lung by positive pressure. A lung protective strategy of limiting tidal volume and plateau pressure is not required for type L but should be actively implemented in type H. However, it is not easy to distinguish between the two types because the lung weight cannot be measured clinically, and the method for evaluating recruitability is unclear. Measuring ventilation-perfusion matching at bedside has not been established. PEEP titration using EIT can not only evaluate recruitability but also determine the optimum PEEP at the time of measurement and provide regional ventilation homogeneity. As a result, it is considered possible to attenuate the regional dynamic strain and inhomogeneity of transpulmonary pressure.There are two phenotypes that have a decisive effect on mechanical ventilation strategies in COVID-19 pneumonia (10) presented three cases of COVID-19 AHRF that had similar levels of oxygenation but variable respiratory system compliance. EIT indicated different characteristics of the regional ventilation profile and was helpful in understanding the etiology of hypoxemia at bedside. Tomasino and colleagues (11) used EIT to identify the characteristics of COVID-19 pneumonia and to decide whether to use high PEEP or prone positioning.To assess the phenotype, regional ventilation distribution was analyzed using EIT in mechanically ventilated patients with acute hypoxemic respiratory failure (AHRF) and ARDS due to COVID-19. Morais and colleagues (12).In a cohort study of mechanically ventilated non-COVID-19 patients, EIT was used to assess the optimal PEEP at which both overdistention and collapse are minimized because both phenomena were equally harmful for ARDS lungs (13) compared the response to PEEP titration between COVID-19 associated ARDS and ARDS from other causes. They defined optimal PEEP using EIT as the smallest sum of overdistention and collapse. COVID-19-associated ARDS lungs required 12 cmH2O of PEEP and had more collapse at low PEEP levels. Van der Zee and colleagues (14) reported that EIT monitoring during the decremental PEEP trial to seek the level of lowest relative alveolar overdistention and collapse provided personalized PEEP that did not result in high driving pressure or transpulmonary pressure. Shono and colleagues personalized PEEP levels and applied prone positioning sessions to obtain homogeneous ventilation distribution based on EIT analysis (15). PEEPset corresponded better with the higher PEEP-FIO2 table (used in ALVEOLI study) than the lower PEEP-FIO2 table (used in ARMA study), especially in patients with a higher body mass index. However, Sella and colleagues (16) compared EIT-guided PEEP and lower PEEP-FIO2 table-guided PEEP and found a correlation. The researchers mentioned that the opposite results might be due to differences in the interpretation of the results presented by the same device and differences in the degree of obesity.Perier and colleagues In addition, marked hypoxemia is often observed even in cases without dorsal collapsed areas in COVID-19-infected lungs. Ventilation-perfusion mismatch is considered to be the major cause of this phenomenon. It is believed that COVID-19 hypercoagulability and vascular endothelial cell damage are responsible for this phenomenon, but the details are unclear. Because ventilation-perfusion mismatch cannot be improved by mechanical ventilation, responding to hypoxemia by increasing ventilation pressure, including PEEP, results in considerable lung injury risk due to overdistention. In summary, investigation of ventilation-perfusion matching on the bedside by EIT is useful for lung protection.(4). Mauri and colleagues reported that the mismatching rate was elevated to 34% (32%-45%) of lung units in COVID-19 lungs (17). Furthermore, in six of seven patients, ventilated nonperfused units (dead space) represented a much larger proportion than perfused nonventilated perfused units (shunt), although the potential for lung recruitment had large variability. The researchers concluded that an elevated dead space fraction might be a specific pathophysiological characteristic of COVID-19 ARDS.Recently, EIT has been used to assess regional lung perfusion distribution in a clinical setting. Rapid injection of a contrast agent such as 10 ml of hypertonic sodium chloride enables the generation of a lung perfusion profile during an end-expiratory hold maneuver Strong inspiratory effort is often observed as a characteristic of COVID-19 pneumonia. Damaged lungs with reduced compliance generate large negative pressure in the thorax, increase transpulmonary pressure, and can cause or aggravate lung injury, patient self-inflicted lung injury (P-SILI). The pathological changes caused by P-SILI are irreversible and worsen the prognosis. Neuromuscular blockade to attenuate inspiratory drive is effective in protecting lungs against P-SILI. However, the use of neuromuscular blockade leads to alveolar collapse, especially in the dorsal regions, with inappropriate PEEP levels, resulting in inhomogeneous ventilation distribution. The efficacy of EIT to prevent P-SILI has not been tested. Further studies are warranted.This review briefly described the basic principle of EIT and summarized the latest findings on its potential contribution to lung protective strategies in COVID-19 patients. EIT is noninvasive and can be used for real-time evaluation to identify the ventilation distribution of infected lungs. The latest approach indicated the feasibility of detecting the distribution of pulmonary blood flow and evaluating ventilation-perfusion matching at bedside.NoneConcept and design: TK. Drafting of the manuscript: TK, AS. The authors read and approved the final manuscript.


Retroperitoneal paragangliomas are rare tumors, they arise from ganglia along the sympathetic and parasympathetic chain. We report a rare case of a non functional paraganglioma in whom surgical resection was performed.A 35 years-old female presented with chronic abdominal pain, A contrast magnetic resonance imaging (MRI) of abdomen showed a well-defined Left latero-aortic cystic retro-peritoneal surgical resection using laparotomy was performed, The patiente recovered well and was discharged three days after surgery. Histological examination and immunohistochemical revealed a retroperitoneal paraganglioma.Non-functioning retroperitoneal paragangliomas are rare and are most often Isolated. Radiological techniques including, Contrast-enhanced computed tomography (CT) and Magnetic resonance imaging (MRI) are useful for identifying and locating retroperitoneal paragangliomas. surgical excision is still the most effective treatment when it possible. •We report a case of non-functional retroperitoneal paraganglioma.•Non-functional retroperitoneal paraganglioma is extremely rare and mostly benign.•A difficult tumour to diagnose and treat.•Complete surgical excision is the treatment of choice for extra-adrenal paragangliomas. Most of them are functional, with symptoms and signs of catecholamine overproduction, similar to pheochromocytoma except for the variation in the anatomic location The risk of malignancy may be as high as 20%–30% compared with a 10% risk of malignancy for adrenal pheochromocytoma [We report a rare case of a non functional paraganglioma in whom surgical resection was performed. This work has been reported in line with the SCARE criteria .2A 35 years-old female with a medical history of chronic abdominal pain for about four months was admitted to our hospital Abdominal examination was normal.Abdominal and pelvis Ultrasound (US) revealed a Left retro peritoneal formation measuring 4cm rounded well-defined with heterogeneous non-vascularized content. A contrast magnetic resonance imaging (MRI) of abdomen showed a well-defined Left latero-aortic cystic retro-peritoneal mass measuring 45 × 50 mm appear as hypointense in T1w and hyperintense in T2w sequence with multiple septa . Biochem3Paraganglioma (PG), also known as extra-adrenal pheochromocytoma, is a chromaffin cell tumor located at various sites along the sympathetic and parasympathetic chain, they can thus be found in the head, neck, thorax, abdomen, and pelvis. Retroperitoneal paragangliomas are rare tumors. Non-functioning retroperitoneal forms are even more rare and are most often isolated .Histologically, most are composed of well-differentiated neuroendocrine cells disposed in small clusters  or cords separated by prominent fibrovascular stroma. Distinctive within the cells in most tumors are dark neurosecretory granules that contain catecholamines. Sometimes the cells are spindle shaped. Mitoses are usually infrequent, but occasional tumors are overtly anaplastic and pleomorphic and contain numerous mitoses. The malignant potential is determined by local invasion and also distant metastases .Functional PGs can be diagnosed based on clinical presentation, the most common type of presenting symptom were associated with catecholamine-secreting paragangliomas: Episodic headache, diaphoresis and tachycardia. Episodic hypertension has been identified as a characteristic feature of catecholamine-secreting paragangliomas and is used for the differential diagnosis of paragangliomas, laboratory examination showing elevated catecholamines and their metabolites (VMA) in the blood and urine. Nonfunctional PGLs are mostly found incidentally or through symptoms caused by compression of the surrounding organs . In our Radiological techniques including Ultrasound, Contrast-enhanced computed tomography (CT) and Magnetic resonance imaging (MRI) are useful for identifying and locating retroperitoneal paragangliomas, the imaging characteristics of retroperitoneal paragangliomas included soft-tissue masses in the sympathetic chains associated with the abdominal aorta, cystic degeneration and necrosis inside the masses functional imaging, such as PET scanning and MIBG scintigraphy may help detect primary or metastatic tumors that could be missed on CT/MRI , in our A complete surgical resection is still the most effective and the only potentially curative treatment and should be attempt even in patients with distant metastasis.Open exploration and resection is the standard surgical management of PG, however, laparoscopic resection of PG is considered challenging because of the altered anatomic location, dense peritumoral adhesions, high vascularity envelope, proximity to major blood vessels ,7. If a Patient with metastatic disease will require adjuvant radiotherapy while chemotherapy is restricted to patients not accessible for surgery and resistant to radionuclide therapy .4Non-functional retroperitoneal paragangliomas are rare group of tumors which are difficult to diagnose, owing to their silent and asymptomatic behavior, Once the diagnosis of a retroperitoneal paraganglioma has been made, surgical excision is required and is still the only curative approach.I declare on my honor that the ethical approval has been exempted by my establishment.None.Amal Hajri: writing the paper.Ahmed Ballati: Corresponding author writing the paper.Zakaria Essaidi: writing the paper.Driss Errguibi: study concept.Rachid Boufettal: study concept.Saad Rifki El Jai: correction of the paper.Farid Chehab: correction of the paper.Written informed consent for publication of their clinical details and/or clinical images was obtained from the patient.Not applicable.DOCTEUR AHMED BALLATI.None.Not commissioned, externally peer-reviewed.The authors declare having no conflicts of interest for this article.


A 69-year-old male presented with medically refractory vertebrobasilar insufficiency and paroxysmal subjective dizziness for six months. Severe stenosis of a dominant left V2 vertebral artery segment was identified on digital subtraction angiography (DSA) with an irregular intraluminal filling defect immediately above the stenosis. Optical coherence tomography (OCT) demonstrated a normal lumen at the distal end, with red thrombus detected distal to the stenosis. Atherosclerotic plaque containing fibro-lipid was also identified and treated with a drug-eluting stent. Distal red thrombi were not covered by stenting, indicating embolization risk in the future. Clear posterior fossa symptoms occurred after intervention, and treatment with a standard dual antiplatelet regimen and statin therapy was prescribed for one year. Six months after treatment, the symptoms improved, and six-minute walking distances were successful with no gait impairment. To our knowledge, this is the first V2 segment stenosis assessed by OCT imaging before and after stenting, indicating an intact fibrous cap with thrombus formation, as well as plaque erosion. Understanding the role and careful use of OCT may improve the identification of red thrombus and plaque erosion when clinically indicated. A 69-year-old male presented with medically refractory vertebrobasilar insufficiency and paroxysmal subjective dizziness for six months. The patient had a history of alcohol use and cigarette smoking for more than 40 years, a four-year history of well-controlled essential hypertension, and a one-month history of documented hyperlipemia. No prior stroke, transient ischemic attack (TIA), myocardial infarction, or significant family history was reported. There was no history of anticoagulation or antiplatelet use. The patient presented with an unremarkable neurological examination. Severe stenosis of the dominant left vertebral artery V2 segment was identified on computed tomography angiography (CTA), likely secondary to atherosclerosis, coexisting with an undeveloped left posterior cerebral artery (PCA) and leading to an incomplete circle of Willis, which was noted A–D. DigiOptical coherence tomography (OCT) is an intravascular imaging method that acquires images at a resolution of ~10 μm, and captures images with an axial resolution 10 times higher than intravascular ultrasound, enabling visualization of the blood vessel wall at near histological resolution [All patients at our center who undergo stenting are pretreated with aspirin (100 mg/d) and clopidogrel (150 mg followed by 75 mg daily). Neurological assessment was performed before and after the procedure. The patients who undergo OCT sign explicit consent. OCT studies are conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Xuanwu Hospital (protocol code (20200913) and date of approval: 30 September 2020. Prior to stenting, standard guidewire placement via a percutaneous puncture of the right femoral artery is performed. A Dragonfly Duo  OCT imaging catheter is placed prior to stenting in order to determine stent sizing, and OCT images in this case confirmed severe stenosis with atherosclerotic plaque formation, as well as fresh mural thrombus (i.e. red thrombus) at the distal end of the stenosis, which has not otherwise been clearly identified. The OCT images demonstrated that this as an irregularly high signal area into the lumen, with hypo-reflective signal attenuation in addition to a fibro-lipid plaque component B–E. A 4.The incidence of red thrombus embolism with distal embolization is high, and possesses a considerable mortality rate, but timely diagnosis is uncommon, and is usually arrived upon after a stroke has occurred. Ideally, this diagnosis should be made at the time of intervention if possible to avoid complications. While DSA has been considered an effective method for visualizing thrombus and embolism as filling defects, the structure of the lumen and the composition of thrombi is relatively poorly resolved. By contrast, OCT is an optical imaging technology characterized by real-time, dynamic intraluminal imaging and high resolution, and very much is its nascence within intracranial vasculature. The OCT imaging guidewire can be passed into distal small vessels for imaging, and OCT images have the ability to clearly depict the luminal structure without neurological complications and identify missed thrombi on DSA, as well as distinguish different types of thrombi, which may guide anticoagulant therapy . OCT and


Mitochondrial uncoupling protein 1 (UCP1) is the crucial mechanistic component of heat production in classical brown fat and the newly identified beige or brite fat. Thermogenesis inevitably comes at a high energetic cost and brown fat, ultimately, is an energy-wasting organ. A constrained strategy that minimizes brown fat activity unless obligate will have been favored during natural selection to safeguard metabolic thriftiness. Accordingly, UCP1 is constitutively inhibited and is inherently not leaky without activation. It follows that increasing brown adipocyte number or UCP1 abundance genetically or pharmacologically does not lead to an automatic increase in thermogenesis or subsequent metabolic consequences in the absence of a plausible route of concomitant activation. Despite its apparent obviousness, this tenet is frequently ignored. Consequently, incorrect conclusions are often drawn from increased BAT or brite/beige depot mass, e.g., predicting or causally linking beneficial metabolic effects. Here, we highlight the inherently inactive nature of UCP1, with a particular emphasis on the molecular brakes and releases of UCP1 activation under physiological conditions. These controls of UCP1 activity represent potential targets of therapeutic interventions to unlock constraints and efficiently harness the energy-expending potential of brown fat to prevent and treat obesity and associated metabolic disorders. Brown and brite/beige adipose tissues are thermogenic and canonically mediate heat generation through mitochondrial respiration that is uncoupled from ATP synthesis via uncoupling protein 1 (UCP1)-mediated proton leak . ThermogIn order to appreciate the role of BAT in overall energy expenditure, and thus in the altered energy balance that leads to obesity and associated diseases, it must be realized that BAT is ultimately an energy-wasting organ and thermogenesis comes at a high energetic cost. While this may appear beneficial in the context of widespread metabolic disease today, it most certainly acted as a constraint during evolutionary time spans of food scarcity, rendering UCP1 inherently inactive when not explicitly required and activated and tightly controlled by multiple regulatory layers, including the transcriptional level, mRNA stability and protein degradation, ensuring cell specificity and temporal control. Consequently, simply increasing BAT mass or UCP1 abundance does not lead to an increase in thermogenesis without a concomitant increase or at least maintenance of the sympathetic nervous system (SNS) tone within BAT or alternative activating stimuli ,7. Thus,It is clear from this derivation that to efficiently harness the energy-converting potential of BAT for the prevention and treatment of obesity and diabetes requires not only knowledge of the substrates and metabolic pathways involved, of the control of these pathways by neurotransmitters and hormones and of the control of gene expression in BAT, but also an appreciation and understanding of the inherently inactive nature of UCP1, the physiological and molecular brakes of UCP1 activation and the exquisite sensitivity of UCP1 activity to temperature-dependent regulation. In this review, we highlight the inherently inactive nature of UCP1, with a particular emphasis on the molecular brakes of UCP1 activation under physiological conditions. We propose that these brakes on UCP1 activity represent potential targets of therapeutic interventions to unlock constraints and efficiently and fully harness the energy-converting potential of brown fat to prevent and treat obesity and associated metabolic disorders.Thermogenesis in brown fat is under tight, unconscious sympathetic control and inherently inactive at the molecular level. The crucial hub of regulation is formed by the functional core of the thermogenic machinery itself, mitochondrial UCP1 and its molecular activity. When active, UCP1 mediates a net proton translocation from the intermembrane space into the mitochondrial matrix, thus diminishing proton motive force in the absence of ADP phosphorylation. The exact molecular mechanism is still under debate . AccumulIt has long been debated whether UCP1 contributes to basal proton conductance of brown fat mitochondria in the absence of adrenergic or other lipolytic stimuli ,13. ThisNotably, comparing UCP1-deficient mitochondria and wildtype controls, no difference in basal proton conductance was observed measuring the overall proton leak kinetics . MoreoveLastly, there are no differences in basal metabolic rate between mice with and without UCP1 ,18. ThisBrown adipose tissue and mitochondrial UCP1 residing therein needs to be activated to produce heat, namely, by free fatty acids liberated through lipolysis. Within minutes of cold exposure, thermal afferent signals elicited by thermoreceptors in the skin are transmitted to the brain, excite the sympathetic nervous system, stimulate sympathetic outflow to BAT and noradrenaline secreted by neuronal varicosities interacts with adrenergic receptors on brown adipocytes. Intracellularly, lipolysis is induced through the canonical adrenergic receptor–Gs-protein–adenylyl cyclase–cAMP–protein kinase A (PKA) pathway. Free fatty acids released from lipid droplets act as both fuel for mitochondrial β-oxidation and activators of UCP1  unequivocally does precisely that, as is verifiable at all levels from isolated mitochondria and cultured cells to brown adipose tissue depots in mice and men.The best-studied scenario of BAT recruitment is undoubtedly the most obvious one, namely, cold exposure. In this condition, activation of thermogenesis and recruitment of thermogenic capacity occurs as a consequence of the same signal: sympathetic stimulation, i.e., norepinephrine release within the tissue . ImportaOne such alternative, potent and fully competent mechanism for BAT recruitment is the PPARγ pathway, probably responsible for the recruitment during pre-hibernation and in the prenatal state. In fact, continuous treatment with the PPARγ agonist rosiglitazone robustly increases the amount of thermogenically competent, UCP1-expressing adipocytes, which are capable of responding to adrenergic stimulation with a dramatic increase in UCP1-mediated oxygen consumption ,39. Despnot thermogenically activated at the same time. Figuratively, BAT recruited in this way represents a larger stove waiting for the spark, an adrenergic stimulus, to initiate heat production. The same rationale must certainly apply to pharmacological or genetic or any other artificial increase in BAT mass or UCP1 abundance. In summary, while doubt remains as to the mechanisms, there clearly are physiological conditions in which brown fat capacity needs to be recruited but, importantly, In the preceding parts, we argued that the amount of UCP1 in brown adipose tissue depots—natural or manipulated—is not a measure of thermogenic activity due to the inherently inactive nature of UCP1 in the absence of an adrenergic stimulus. An obvious conclusion then appears to activate brown fat by norepinephrine or synthetic, adrenergic agonists to alleviate this concern . This ceIn cultured brown adipocytes, for instance, UCP1 abundance will not increase baseline activation (see above). At the same time, stimulated full activation does also not allow additional conclusions beyond the functional capacity of the cell model. In vivo, accordingly, the classical norepinephrine test—injection of norepinephrine/CL-316243 during indirect calorimetry—will only allow the quantification of thermogenic capacity, not the contribution of any amount of brown fat or UCP1 to metabolism under free-running conditions.Acute thermogenic power is necessarily tightly controlled and subject to feedback mechanisms. In essence, the amount of heat produced is regulated to match the amount of heat lost, to preserve normothermia. A very vivid example is the production of extra heat by treating animals with the chemical uncoupler DNP. This intervention leads to a robust increase in metabolic rate at thermoneutrality but fails to do so at colder ambient temperature  because One way to circumvent this conundrum is to directly monitor brown fat thermogenesis in real-time using thermosensors that are implanted in proximity to interscapular BAT under free-running conditions . This phCollectively, physiological changes in response to a manipulated UCP1 abundance cannot be causally attributed to endogenous, UCP1-dependent thermogenesis, neither by indirect calorimetry in vivo nor by cellular respirometry, neither in a basal state nor activated by cold or norepinephrine.abundance beyond thermogenic capacity by activating thermogenesis. However, one can certainly determine such consequences of altered UCP1 activity.We are unable to determine physiological consequences of altered UCP1 Thermogenesis inevitably comes at a high energetic cost. The full activation of UCP1-mediated energy-dissipating mechanisms can contribute to whole body energy substrate homeostasis, exert significant metabolic benefits and protect from a chronic imbalance between energy intake and expenditure. This concept emerged in the late 1970s and resulted from two key observations : (1) feeNevertheless, there is considerable controversy regarding the contribution of brown fat to prevent the development of diet-induced obesity, even though a recruitment of UCP1 in response to HFD feeding has often been reported . The mosper se does not protect mice from diet-induced obesity under physiological conditions, irrespective of the type of diet or the ambient temperature. Pharmacological activation of UCP1, however, by daily injection of the selective β3-adrenergic receptor agonist CL316,243, resulted in a significant reduction of body weight in a UCP1-dependent manner [In summary, UCP1 t manner . In factt manner . Therefo®) caused enhanced energy expenditure in humans  [In humans, the anti-obesity and metabolic efficacy of the β3-adrenergic agonist CL316243 is limited for multiple reasons, such as low efficacy at the human β3AR, low β3AR expression levels, a limited amount of brown fat and cardiovascular side-effects. The β3-specific agonist mirabegron  . Howevercal/day)  or β2 adcal/day) . Moreovecal/day) . Thus, aThe inherently inactive nature of UCP1 impedes quantification of its contribution to energy balance and complicates the pharmacological targeting of UCP1 abundance and brown fat cell number. On the other hand, negative regulators of thermogenic activity may serve as a largely unexplored and unexploited resource of novel target mechanisms.In the broadest physiological sense, brown fat activity is negatively regulated by any environmental, allometric or behavioral condition that causes reduced thermogenic demand . This inAt a given temperature gradient, heat flow is a function of insulation and interface surface. The former is a highly relevant determinant of thermogenic demand, as demonstrated in mouse models of furlessness and skin barrier defects ,74,75. TFinally, thermogenesis comes at a high energetic cost. When there is insufficient energy available, as in response to fasting, thermogenesis is severely impaired to conserve energy. The fasting-induced reduction in thermogenic capacity is linked to thyroid hormone levels  and BAT Together, these phenomena form another major hurdle for brown fat-based therapeutic strategies, beyond the limited amount of human brown fat. Modern humans spend much of their time at thermoneutrality, possibly explaining a low brown fat amount and capacity in humans. Furthermore, patients benefiting most from a novel treatment for metabolic disease will typically be of larger than average body mass. In the following, we review the neural, endocrine and molecular mechanisms underlying suppression of BAT activity. All of them constitute potential constraints on future BAT-centric therapies and at the same time, and for the same reason, promising targets for complementary intervention.BAT is highly innervated by the sympathetic nervous system and contains a rich vascular supply . Both chSympathetic tone is determined by two key parameters: norepinephrine (NE) release, a direct function of sympathetic activity, and extracellular NE clearance, which is key to terminating adrenergic signaling. Historically, NE clearance was believed to occur exclusively through active transport via the monoamine transporter SLC6A2 of SNS nerve terminals . After cA well-established paradigm for brown fat activation is activation of Gs protein-coupled receptors (GPCRs) initiating cAMP generation . Vice veThe second messengers cAMP and cGMP are central for thermogenic activation of brown adipocytes . PhosphoPKA-mediated lipolysis activation represents the canonical pathway to stimulate thermogenesis. When inactive, PKA is a tetrameric holoenzyme comprising two regulatory (R) subunits and two catalytic (C) subunits . BindingUltimately, cessation of lipolysis depends on the dephosphorylation of PKA targets, such as HSL and perilipin, by protein phosphatases; therefore, cAMP degradation and PKA inactivation do not per se terminate the lipolytic cascade and thermogenesis . ThroughUCP1, the central mechanistic component of brown fat-mediated non-shivering thermogenesis, is itself a regulatory center inhibited by purine nucleotides  . Purine Our understanding of the physiological functions of BAT has continuously evolved. It is now evident that it fulfils multiple important roles beyond the core of thermoregulation. The energy-expending and metabolic sink properties of activated BAT make it an attractive target for combating obesity and metabolic disease. However, brown fat is ultimately an energy-wasting organ and under tight control. Consequently, brown fat thermogenesis is highly sensitive to changes in ambient temperature and is only activated by heat loss, less active when temperature rises and even repressed when ambient temperature reaches the thermoneutral zone. Since humans usually live in an environment close to thermoneutrality, this represents a major hurdle for the development of brown fat-based therapeutic strategies against metabolic disorders, regardless of whether BAT is present in sufficient amounts. In short, due to built-in brake systems, UCP1 is inherently inactive and does not produce heat without activation. Therefore, understanding the brake systems on brown fat thermogenesis may reveal novel targets of therapeutic intervention to unlock constraints and efficiently harness the energy-expending potential of brown fat.