Plasma Proteomics Identify Biomarkers and Pathogenesis of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is a global public health crisis. However, little is known about the pathogenesis and biomarkers of COVID-19. Here, we profiled host responses to COVID-19 by performing plasma proteomics of a cohort of COVID-19 patients, including non-survivors and survivors recovered from mild or severe symptoms, and uncovered numerous COVID-19-associated alterations of plasma proteins. We developed a machine-learning-based pipeline to identify 11 proteins as biomarkers and a set of biomarker combinations, which were validated by an independent cohort and accurately distinguished and predicted COVID-19 outcomes. Some of the biomarkers were further validated by enzyme-linked immunosorbent assay (ELISA) using a larger cohort. These markedly altered proteins, including the biomarkers, mediate pathophysiological pathways, such as immune or inflammatory responses, platelet degranulation and coagulation, and metabolism, that likely contribute to the pathogenesis. Our findings provide valuable knowledge about COVID-19 biomarkers and shed light on the pathogenesis and potential therapeutic targets of COVID-19.

Plasma metabolome and cytokine profile reveal glycylproline modulating antibody fading in convalescent COVID-19 patients.

The COVID-19 pandemic has incurred tremendous costs worldwide and is still threatening public health in the "new normal." The association between neutralizing antibody levels and metabolic alterations in convalescent patients with COVID-19 is still poorly understood. In the present work, we conducted absolutely quantitative profiling to compare the plasma cytokines and metabolome of ordinary convalescent patients with antibodies (CA), convalescents with rapidly faded antibodies (CO), and healthy subjects. As a result, we identified that cytokines such as M-CSF and IL-12p40 and plasma metabolites such as glycylproline (gly-pro) and long-chain acylcarnitines could be associated with antibody fading in COVID-19 convalescent patients. Following feature selection, we built machine-learning-based classification models using 17 features (six cytokines and 11 metabolites). Overall accuracies of more than 90% were attained in at least six machine-learning models. Of note, the dipeptide gly-pro, a product of enzymatic peptide cleavage catalyzed by dipeptidyl peptidase 4 (DPP4), strongly accumulated in CO individuals compared with the CA group. Furthermore, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination experiments in healthy mice demonstrated that supplementation of gly-pro down-regulates SARS-CoV-2-specific receptor-binding domain antibody levels and suppresses immune responses, whereas the DPP4 inhibitor sitagliptin can counteract the inhibitory effects of gly-pro upon SARS-CoV-2 vaccination. Our findings not only reveal the important role of gly-pro in the immune responses to SARS-CoV-2 infection but also indicate a possible mechanism underlying the beneficial outcomes of treatment with DPP4 inhibitors in convalescent COVID-19 patients, shedding light on therapeutic and vaccination strategies against COVID-19.

Changes in Diversity and Composition of Rhizosphere Bacterial and Fungal Community between Resistant and Susceptible Pakchoi under Plasmodiophora brassicae.

Plasmodiophora brassicae (P. brassicae) is a soil-born pathogen worldwide and can infect most cruciferous plants, which causes great yield decline and economic losses. It is not well known how microbial diversity and community composition change during P. brassicae infecting plant roots. Here, we employed a resistant and a susceptible pakchoi cultivar with and without inoculation with P. brassicae to analyze bacterial and fungal diversity using 16S rRNA V3-V4 and ITS_V1 regions, respectively. 16S rRNA V3-V4 and ITS_V1 regions were amplified and sequenced separately. Results revealed that both fungal and bacterial diversity increased, and composition was changed in the rhizosphere soil of the susceptible pakchoi compared with the resistant cultivar. In the four groups of R_mock, S_mock, R_10d, and S_10d, the most relatively abundant bacterium and fungus was Proteobacteria, accounting for 61.92%, 58.17%, 48.64%, and 50.00%, respectively, and Ascomycota, accounting for 75.11%, 63.69%, 72.10%, and 90.31%, respectively. A total of 9488 and 11,914 bacteria were observed uniquely in the rhizosphere soil of resistant and susceptible pakchoi, respectively, while only 80 and 103 fungi were observed uniquely in the correlated soil. LefSe analysis showed that 107 and 49 differentially abundant taxa were observed in bacteria and fungi. Overall, we concluded that different pakchoi cultivars affect microbial diversity and community composition, and microorganisms prefer to gather around the rhizosphere of susceptible pakchoi. These findings provide a new insight into plant-microorganism interactions.

Extracorporeal membrane oxygenation for avian influenza A (H7N9) patient with acute respiratory distress syndrome: a case report and short literature review.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is performed as an acceptable life-saving bridging procedure in patients with severe acute respiratory distress syndrome (ARDS).To patients with avian influenza A (H7N9)-associated ARDS, ECMO could be adopted as a feasible therapeutic solution. We present our successful experience with ECMO utilized in a respiratory failure patient with H7N9 infection. CASE PRESENTATION: A 44 years-old female with H7N9-induced ARDS was admitted to intensive care unit (ICU) and was treated with veno-venous ECMO for six days, antiviral therapy, prolonged corticosteroid infusion and other therapies. She suffered significant hemorrhage requiring transfusion of platelets and multidrug-resistant Acinetobacter Baumannii infection during ECMO support. Bleeding and infection almost killed the patient's life. Fortunately, she was alive at last and completly recovered after 38 days of ICU stay. CONCLUSIONS: ECMO was effective in this H7N9 patient with a fatal respiratory failure. Mechanical circulatory support was the only chance for our patient with H7N9-associated ARDS to survive until respiratory function recovery. Early detection and rapid response are essential to these serious ECMO-associated complications such as hemorrhage, thrombosis and infection.

MiR-141 targets ZEB2 to suppress HCC progression.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Increasing evidence suggests that microRNAs (miRNAs) are associated with HCC tumorigenesis. The present study was designed to define the role of miR-141 in HCC. The expression of miR-141 was significantly decreased in four HCC cell lines. Overexpression of miR-141 suppressed both the growth and the motility of HCC cells. Furthermore, we identified zinc finger E-box binding homeobox 2 (ZEB2) as a target of miR-141 and miR-141 functioned as a tumor suppressor via ZEB2 targeting in HCC. These data provide a novel potential therapeutic target for HCC treatment.

Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a.

CRISPR-based assays for the detection of nucleic acids are highly specific, yet they are not fast, sensitive or easy to use. Here we report a one-step fluorescence assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal samples, with a sample-to-answer time of less than 20 minutes and a sensitivity comparable to that of quantitative real-time PCR with reverse transcription (RT-qPCR). The assay uses suboptimal protospacer adjacent motifs, allowing for flexibility in the design of CRISPR RNAs and slowing down the kinetics of Cas12a-mediated collateral cleavage of fluorescent DNA reporters and cis cleavage of substrates, which leads to stronger fluorescence owing to the accumulation of amplicons generated by isothermal recombinase polymerase amplification. In a set of 204 nasopharyngeal samples with RT-qPCR cycle thresholds ranging from 18.1 to 35.8, the assay detected SARS-CoV-2 with a sensitivity of 94.2% and a specificity of 100%, without the need for RNA extraction. Rapid and sensitive assays for nucleic acid testing in one pot that allow for flexibility in assay design may aid the development of reliable point-of-care nucleic acid testing.

Recent Advances in Bunyavirus Reverse Genetics Research: Systems Development, Applications, and Future Perspectives.

Bunyaviruses are members of the Bunyavirales order, which is the largest group of RNA viruses, comprising 12 families, including a large group of emerging and re-emerging viruses. These viruses can infect a wide variety of species worldwide, such as arthropods, protozoans, plants, animals, and humans, and pose substantial threats to the public. In view of the fact that a better understanding of the life cycle of a highly pathogenic virus is often a precondition for developing vaccines and antivirals, it is urgent to develop powerful tools to unravel the molecular basis of the pathogenesis. However, biosafety level -3 or even -4 containment laboratory is considered as a necessary condition for working with a number of bunyaviruses, which has hampered various studies. Reverse genetics systems, including minigenome (MG), infectious virus-like particle (iVLP), and infectious full-length clone (IFLC) systems, are capable of recapitulating some or all steps of the viral replication cycle; among these, the MG and iVLP systems have been very convenient and effective tools, allowing researchers to manipulate the genome segments of pathogenic viruses at lower biocontainment to investigate the viral genome transcription, replication, virus entry, and budding. The IFLC system is generally developed based on the MG or iVLP systems, which have facilitated the generation of recombinant infectious viruses. The MG, iVLP, and IFLC systems have been successfully developed for some important bunyaviruses and have been widely employed as powerful tools to investigate the viral replication cycle, virus-host interactions, virus pathogenesis, and virus evolutionary process. The majority of bunyaviruses is generally enveloped negative-strand RNA viruses with two to six genome segments, of which the viruses with bipartite and tripartite genome segments have mostly been characterized. This review aimed to summarize current knowledge on reverse genetic studies of representative bunyaviruses causing severe diseases in humans and animals, which will contribute to the better understanding of the bunyavirus replication cycle and provide some hints for developing designed antivirals.

Non-structural Proteins of Severe Fever With Thrombocytopenia Syndrome Virus Suppress RNA Synthesis in a Transcriptionally Active cDNA-Derived Viral RNA Synthesis System.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the tick-borne SFTS bunyavirus (SFTSV) resulting in a high fatality rate up to 30%. SFTSV is a negative-strand RNA virus containing three single-stranded RNA genome segments designated as L, M, and S, which respectively, encode the RNA-dependent RNA polymerase (RdRp), glycoproteins Gn and Gc, and nucleoprotein (N) and non-structural proteins (NSs). NSs can form inclusion bodies (IBs) in infected and transfected cells. A previous study has provided a clue that SFTSV NSs may be involved in virus-like or viral RNA synthesis; however, the details remain unclear. Our work described here reveals that SFTSV NSs can downregulate virus-like RNA synthesis in a dose-dependent manner within a cDNA-derived viral RNA synthesis system, i.e., minigenome (-) and minigenome (+) systems based on transfection, superinfection, and luciferase reporter activity determination; meanwhile, NSs also show a weak inhibitory effect on virus replication. By using co-immunoprecipitation (Co-IP) and RT-PCR combined with site-directed mutagenesis, we found that NSs suppress virus-like RNA or virus replication through interacting with N but not with RdRp, and the negative regulatory effect correlates closely with the IB structure it formed but is not associated with its role of antagonizing host innate immune responses. When the cytoplasmic structure of IB formed by SFTSV NSs was deprived, the inhibitory effect of NSs on virus-like RNA synthesis would weaken and even disappear. Similarly, we also evaluated other bandavirus NSs that cannot form IB in neither infected nor transfected cells, and the results showed that the NSs of Heartland bandavirus (HRTV) did not show a significant inhibitory effect on virus-like RNA synthesis within a minigenome system. Our findings provide experimental evidence that SFTSV NSs participate in regulating virus-like or viral RNA synthesis and the negative effect may be due to the NSs-N interaction.

Infection-associated Hemophagocytic Syndrome in Critically Ill Patients with COVID-19.

Infection-associated hemophagocytic syndrome (IAHS), a severe complication of various infections, is potentially fatal. This study aims to determine whether IAHS occurs in critically ill patients with coronavirus disease 2019 (COVID-19). We conducted a retrospective observational study on 268 critically ill patients with COVID-19 between February 1st, 2020 and February 26th, 2020. Demographics, clinical characteristics, laboratory results, information on concurrent treatments and outcomes were collected. A diagnosis of secondary hemophagocytic lymphohistiocytosis (sHLH) was made when the patients had an HScore greater than 169. Histopathological examinations were performed to confirm the presence of hemophagocytosis. Of 268 critically ill patients with confirmed SARS-CoV-2 infection, 17 (6.3%) patients had an HScore greater than 169. All the 17 patients with sHLH died. The interval from the onset of symptom of COVID-19 to the time of a diagnosis of sHLH made was 19 days and the interval from the diagnosis of sHLH to death was 4 days. Ten (59%) patients were infected with only SARS-CoV-2. Hemophagocytosis in the spleen and the liver, as well as lymphocyte infiltration in the liver on histopathological examinations, was found in 3 sHLH autopsy patients. Mortality in sHLH patients with COVID-19 is high. And SARS-CoV-2 is a potential trigger for sHLH. Prompt recognition of IAHS in critically ill patients with COVID-19 could be beneficial for improving clinical outcomes.

A cohort autopsy study defines COVID-19 systemic pathogenesis.

Severe COVID-19 disease caused by SARS-CoV-2 is frequently accompanied by dysfunction of the lungs and extrapulmonary organs. However, the organotropism of SARS-CoV-2 and the port of virus entry for systemic dissemination remain largely unknown. We profiled 26 COVID-19 autopsy cases from four cohorts in Wuhan, China, and determined the systemic distribution of SARS-CoV-2. SARS-CoV-2 was detected in the lungs and multiple extrapulmonary organs of critically ill COVID-19 patients up to 67 days after symptom onset. Based on organotropism and pathological features of the patients, COVID-19 was divided into viral intrapulmonary and systemic subtypes. In patients with systemic viral distribution, SARS-CoV-2 was detected in monocytes, macrophages, and vascular endothelia at blood-air barrier, blood-testis barrier, and filtration barrier. Critically ill patients with long disease duration showed decreased pulmonary cell proliferation, reduced viral RNA, and marked fibrosis in the lungs. Permanent SARS-CoV-2 presence and tissue injuries in the lungs and extrapulmonary organs suggest direct viral invasion as a mechanism of pathogenicity in critically ill patients. SARS-CoV-2 may hijack monocytes, macrophages, and vascular endothelia at physiological barriers as the ports of entry for systemic dissemination. Our study thus delineates systemic pathological features of SARS-CoV-2 infection, which sheds light on the development of novel COVID-19 treatment.

SARS-Coronavirus-2 Nsp13 Possesses NTPase and RNA Helicase Activities That Can Be Inhibited by Bismuth Salts.

The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.

Imaging Mass Cytometric Analysis of Postmortem Tissues Reveals Dysregulated Immune Cell and Cytokine Responses in Multiple Organs of COVID-19 Patients.

SARS-coronavirus-2-induced immune dysregulation and inflammatory responses are involved in the pathogenesis of coronavirus disease-2019 (COVID-19). However, very little is known about immune cell and cytokine alterations in specific organs of COVID-19 patients. Here, we evaluated immune cells and cytokines in postmortem tissues, i.e., lungs, intestine, liver, kidneys, and spleen of three patients with COVID-19. Imaging mass cytometry revealed monocyte, macrophage, and dendritic cell (DC) infiltration in the lung, intestine, kidney, and liver tissues. Moreover, in patients with COVID-19, natural killer T cells infiltrated the liver, lungs, and intestine, whereas B cells infiltrated the kidneys, lungs, and intestine. CD11b+ macrophages and CD11c+ DCs also infiltrated the lungs and intestine, a phenomenon that was accompanied by overproduction of the immunosuppressive cytokine interleukin (IL)-10. However, CD11b+ macrophages and CD11c+ DCs in the lungs or intestine of COVID-19 patients did not express human leukocyte antigen DR isotype. In contrast, tumor necrosis factor (TNF)-α expression was higher in the lungs, intestine, liver, and kidneys, but not in the spleen, of all COVID-19 patients (compared to levels in controls). Collectively, these findings suggested that IL-10 and TNF-α as immunosuppressive and pro-inflammatory agents, respectively,-might be prognostic and could serve as therapeutic targets for COVID-19.

Plasma metabolomic and lipidomic alterations associated with COVID-19.

The pandemic of the coronavirus disease 2019 (COVID-19) has become a global public health crisis. The symptoms of COVID-19 range from mild to severe, but the physiological changes associated with COVID-19 are barely understood. In this study, we performed targeted metabolomic and lipidomic analyses of plasma from a cohort of patients with COVID-19 who had experienced different symptoms. We found that metabolite and lipid alterations exhibit apparent correlation with the course of disease in these patients, indicating that the development of COVID-19 affected their whole-body metabolism. In particular, malic acid of the TCA cycle and carbamoyl phosphate of the urea cycle result in altered energy metabolism and hepatic dysfunction, respectively. It should be noted that carbamoyl phosphate is profoundly down-regulated in patients who died compared with patients with mild symptoms. And, more importantly, guanosine monophosphate (GMP), which is mediated not only by GMP synthase but also by CD39 and CD73, is significantly changed between healthy subjects and patients with COVID-19, as well as between the mild and fatal cases. In addition, dyslipidemia was observed in patients with COVID-19. Overall, the disturbed metabolic patterns have been found to align with the progress and severity of COVID-19. This work provides valuable knowledge about plasma biomarkers associated with COVID-19 and potential therapeutic targets, as well as an important resource for further studies of the pathogenesis of COVID-19.

A novel HLA class I allele, HLA-B*46:01:21, identified by sequence-based typing in a Chinese individual.

HLA-B*46:01:21 differs from HLA-B*46:01:01 by one nucleotide exchange at position 834(G>A) with no amino exchange.

Treatment of chronic tuberculous empyema by intrapleural injection of anhydrous ethanol.

BACKGROUND: Anhydrous ethanol, for its part, has been successfully used to treat renal cyst, hepatocellular carcinoma and ovarian chocolate cyst et al. However, in spite of the high frequency of tuberculous purulent pleural effusion, we found that only a few very early studies that attempted to assess the use of intrapleural anhydrous ethanol in tuberculous effusions with signs of empyema. We report a patient who was injected anhydrous ethanol into pleural cavity to treat chronic tuberculous empyema. CASE PRESENTATION: A 23-year old male was admitted in the hospital because of chronic tuberculous empyema. Ultra-sonography guided thoracentesis and thoracic close drainages were done, but had no effect. However, the patient refused Video-assisted Thoracoscopic Surgery (VATS) and traditional thoracotomy. Therefore, we injected anhydrous ethanol into the pleural cavity after getting the patient's consent. Pyothorax was quickly controlled and the patient finally recovered fully. CONCLUSION: Surgical operation is the main treatment of chronic tuberculous empyema, which has a high cost and large injury, and many patients do not accept this treatment. In this study, intrapleural injection of anhydrous ethanol could achieve the purpose of eliminating the pus cavity, which is particularly suitable for patients who cannot tolerate surgery or are unwilling to undergo surgical treatment.

Regulation Mechanism of MicroRNAs in Non-Small Cell Lung Cancer.

MicroRNAs are small non-coding RNAs with regulatory biological activity, by modulating target genes on epigenetic, transcriptional, post-transciptional and translational levels. Hundreds of reports indicated that miRNAs play important roles in non-small cell lung cancer (NSCLC). Actually, microRNAs are both regulation targets and regulators targeting effector genes. This article reviewed multifaceted role of microRNAs associated to NSCLC, not only targeting to but also targeted by tumor related genes, to help us understand microRNAs related complex regulation networks. Aberrant expressed micoRNAs and their targets were summarized; the statistical results showed that several microRNAs may play key roles by targeting multiple tumor associated targets. On the other hand, Oncogenes and tumor repressors represented by PTEN were also shown to be the most popular targets of microRNAs. Additionally, ZEB1/2 may be a featured pathway in NSCLC, with significant frequency modulated by microRNAs.

Alteration of circadian rhythm during epileptogenesis: implications for the suprachiasmatic nucleus circuits.

It is important to realize that characterization of the circadian rhythm patterns of seizure occurrence can implicate in diagnosis and treatment of selected types of epilepsy. Evidence suggests a role for the suprachiasmatic nucleus (SCN) circuits in overall circadian rhythm and seizure susceptibility both in animals and humans. Thus, we conclude that SCN circuits may exert modifying effects on circadian rhythmicity and neuronal excitability during epileptogenesis. SCN circuits will be studied in our brain centre and collaborating centres to explore further the interaction between the circadian rhythm and epileptic seizures. More and thorough research is warranted to provide insight into epileptic seizures with circadian disruption comorbidities such as disorders of cardiovascular parameters and core body temperature circadian rhythms.

Neuroanatomical circuitry between kidney and rostral elements of brain: a virally mediated transsynaptic tracing study in mice.

The identity of higher-order neurons and circuits playing an associative role to control renal function is not well understood. We identified specific neural populations of rostral elements of brain regions that project multisynaptically to the kidneys in 3-6 days after injecting a retrograde tracer pseudorabies virus (PRV)-614 into kidney of 13 adult male C57BL/6J strain mice. PRV-614 infected neurons were detected in a number of mesencephalic (e.g. central amygdala nucleus), telencephalic regions and motor cortex. These divisions included the preoptic area (POA), dorsomedial hypothalamus (DMH), lateral hypothalamus, arcuate nucleus (Arc), suprachiasmatic nucleus (SCN), periventricular hypothalamus (PeH), and rostral and caudal subdivision of the paraventricular nucleus of the hypothalamus (PVN). PRV-614/Tyrosine hydroxylase (TH) double-labeled cells were found within DMH, Arc, SCN, PeH, PVN, the anterodorsal and medial POA. A subset of neurons in PVN that participated in regulating sympathetic outflow to kidney was catecholaminergic or serotonergic. PRV-614 infected neurons within the PVN also contained arginine vasopressin or oxytocin. These data demonstrate the rostral elements of brain innervate the kidney by the neuroanatomical circuitry.

Video-assisted thoracoscopic surgery for the treatment of mediastinal lymph node tuberculous abscesses.

The mediastinal lymph node tuberculous abscesses (MLNTAs) are secondary to mediastinal tuberculous lymphadenitis. Surgical excision is often required when cold abscesses form. This study was aimed to examine video-assisted thoracoscopic surgery (VATS) for the treatment of MLNTA. Clinical data of 16 MLNTA patients who were treated in our hospital between December 1, 2013 and December 1, 2015 were retrospectively analyzed. All of the patients underwent the radical debridement and drainage of abscesses, and intrathoracic lesions were removed by VATS. They were also administered the intensified anti-tuberculosis treatment (ATT), and engaged in normal physical activity and follow-up for 3 to 6 months. The results showed that VATS was successfully attempted in all of the 16 MLNTA patients and they all had good recovery. Two patients developed complications after surgery, with one patient developing recurrent laryngeal nerve injury, and the other reporting poor wound healing. It was concluded that VATS is easy to perform, and safe, and has high rates of success and relatively few side-effects when used to treat MLNTA.

The caudal pedunculopontine tegmental nucleus may be involved in the regulation of skeletal muscle activity by melanocortin-sympathetic pathway: a virally mediated trans-synaptic tracing study in spinally transected transgenic mice.

Understanding neuroanatomical sympathetic circuitry and neuronal connections from the caudal pedunculopontine tegmental nucleus to skeletal muscle is important to the study of possible mechanisms of pedunculopontine tegmental nucleus (PPTg) and cuneiform nucleus (CnF) that are involved in the regulation of skeletal muscle activity of the sympathetic pathway. The aim of this study was to use virus PRV-614 to trace the melanocortin-sympathetic neural pathways from PPTg and CnF to a hindlimb muscle (gastrocnemius) in spinally transected MC4R-GFP transgenic mice. PRV-614 was injected into the gastrocnemius muscle after receiving a complete spinal cord transection below the L2 level. PRV-614/MC4R-GFP and PRV-614/TPH dual-labeled neurons were found in the dissipated parts of PPTg (dpPPTg), but not between the compact parts of PPTg (cpPPTg) and CnF. It is proposed that a hierarchical pathway of neurons within the caudal pedunculopontine tegmental nucleus sends projections to the RVLM, which in turn projects onto the IML sympathetic preganglionic neurons that regulate muscle blood flow through melanocortin-sympathetic signals. Our results collectively indicate that MC4Rs expressed in caudal pedunculopontine tegmental nucleus may be involved in skeletal muscle activity of melanocortin-sympathetic pathways.