Whole parasite vaccines provide a unique opportunity for dissecting immune mechanisms and identify antigens that are targeted by immune responses which have the potential to mediate sterile protection against malaria infections. The radiation attenuated sporozoite (PfSPZ) vaccine has been considered the gold standard for malaria vaccines because of its unparalleled efficacy. The immunogenicity of this and other vaccines continues to be evaluated by using recombinant proteins or peptides of known sporozoite antigens. This approach, however, has significant limitations by relying solely on a limited number of known pathogen-associated immune epitopes. Using the full range of antigens expressed by the sporozoite will enable the comprehensive immune-profiling of humoral immune responses induced by whole parasite vaccines. To address this challenge, a novel ELISA based on sporozoites was developed.The SPZ-ELISA method described in this report can be performed with either freshly dissected sporozoites or with cryopreserved sporozoite lysates. The use of a fixative for reproducible coating is not required. The SPZ-ELISA was first validated using monoclonal antibodies specific for CSP and TRAP and then used for the characterization of immune sera from radiation attenuated sporozoite vaccinees.Applying this simple and highly reproducible approach to assess immune responses induced by malaria vaccines, both recombinant and whole parasite vaccines, (1) will help in the evaluation of immune responses induced by antigenically complex malaria vaccines such as the irradiated SPZ-vaccine, (2) will facilitate and accelerate the identification of immune correlates of protection, and (3) can also be a valuable assessment tool for antigen discovery as well as down-selection of vaccine formulations and, thereby, guide vaccine design.The online version of this article (10.1186/s12936-017-2129-9) contains supplementary material, which is available to authorized users. To date, only whole parasite vaccines have been able to reproducibly induce high vaccine efficacy against malaria infection in humans  (reviewed in ). The quMajor efforts have been undertaken to make the radiation attenuated sporozoite vaccine (PfSPZ) a viable alternative to recombinant vaccines against malaria. The PfSPZ-vaccine is based on the observation that large numbers of infectious bites from irradiated mosquitoes mediate sterile protection. The original approach required the immunization to take place close to a large insectary capable of supplying sufficient numbers of radiation attenuated, infected mosquitoes , 3. More+ T cells, IFN-γ, and antibodies as being essential for providing protection [+ T cells in mediating protection in human vaccinees is less supported by data [Apart from the obvious desire to develop a protective malaria vaccine, whole parasite vaccines also offer the opportunity to study immune mechanisms that lead to sterile protection. These findings can then be used to develop recombinant vaccines that emulate a sporozoite-based vaccine. Early studies with the radiation attenuated sporozoite (RAS) vaccine in preclinical models identified CD8otection . While t by data , they ha by data . This co by data –9.Antibodies induced by whole parasite vaccines are mostly measured using conventional ELISA methods that employ either the full-length circumsporozoite protein (CSP) or the central repeat region of the CSP as plate antigen. While CSP is the major surface protein, using only a single antigen to assess the immunogenicity and magnitude of the vaccine induced humoral immune response provides quite limited insight into the vaccine-induced humoral immune response and may result in flawed conclusions and misdirect the search for immune correlates of protection.Plasmodium falciparum sporozoites as plate antigen. To date, anti-sporozoite serological responses are captured by performing immunofluorescence assays with sporozoites [Plasmodium yoelii sporozoite vaccines where sporozoite lysates were used in an ELISA format [P. falciparum sporozoites as plate antigen in an ELISA. In reports where researchers utilize ELISA assays to detect or measure the number of sporozoites, monoclonal antibodies specific for CSP are used to capture sporozoites in a sandwich ELISA format [This report describes a novel, simple, and highly reproducible ELISA protocol (SPZ-ELISA) based on employing rozoites –12. EvalA format , 14. To A format  and suchSporozoites were prepared by dissecting mosquitoes 16–20 days post blood feed using the Ozaki method [For the detection of sporozoite antigens, the following antibodies were used: 2A10 , clone SAI171C-5E2 , clones TH1 and TH3 , de-identified serum pools from radiation attenuated sporozoite-immunized (RAS) vaccinees [Freshly dissected sporozoites or thawed sporozoite pellets were suspended to the desired concentration with PBS (pH 7.4) and plated at 30 μl/well in Immulon 2HB plates . Plates were incubated for 2 h at room temperature (RT). The liquid was gently removed from wells and plates were either allow to air dry or fixed by adding 50 μl/well fixative . Plates were then blocked with PBS + 1% BSA (50 μl/well) for 1 h at RT. Primary antibodies were diluted with PBS + 1% BSA, added to the respective wells (50 μl/well), and plates were incubated for 2 h at RT. Unbound antibodies were removed by washing the plates three times with PBS. Secondary antibodies were diluted with PBS + 1% BSA (1:200) and added to each well (50 μl/well) for 1 h at RT. Plates were then washed with PBS and substrate  was added. Plates were read on a SpectraMax M2 plate reader  at 630 nm absorption.Statistically significant differences between the various assay conditions were determined by using two-sided T tests .First, the sporozoite concentration for coating was determined for both intact and lysed sporozoites   regardless of the protective status of the vaccinees. When comparing the protected vs. non-protected groups, serum from protected vaccinees generated a significantly stronger signal when using air dried lysates . The reactivity of the protected vs. non-protected serum pools was not as significant when using methanol fixed sporozoite lysates and when coating with the higher antigen concentration; only at the lower SPZ concentration the difference was significant . These results suggest that the sporozoite ELISA may be a valuable tool for the analysis of whole sporozoite vaccines.Lastly, it was determined whether the SPZ-ELISA can be used to distinguish specificities of various CSP-specific monoclonal antibodies, and whether other sporozoite specific antigens such as TRAP can be detected Fig. . While tThe present study describes the development of an experimental approach to assess immune responses induced by malaria vaccines, in particular whole parasite vaccines. The use of reagents that can easily be made available to any laboratory was a major consideration and, therefore, the use of sporozoite lysates was tested regarding its impact on the resulting immunoreactivity in the ELISA method. The method described here represents an asset for the evaluation of immune responses induced by antigenically complex malaria vaccines such as the irradiated SPZ-vaccine and will, therefore, facilitate and accelerate the identification of immune correlates of protection.


There are over 3 million Americans infected with hepatitis C virus (HCV). Despite recent advances in HCV treatment, a major barrier to care remains a limited number of treaters. HCV therapy provision by primary care providers (PCPs) could expand access by increasing the pool of HCV treating clinicians.To characterize current HCV care practices, willingness and self-efficacy of PCPs to become HCV treaters.Two hundred and seventy one PCPs were identified from community clinics affiliated with a large academic center and 4 large federally qualified health centers in Baltimore, MD. An internet-based survey was administered to assess provider demographics, clinical practice site and willingness to provide HCV care. Factors associated with willingness to provide HCV care were examined using odds ratios (OR).Among 129 (48%) PCPs who responded, the majority (70%) had an MD/DO degree and were white (60%). Only a few PCPs, 12 (10%), had treated at least 1 patient for HCV in the prior year. Although only 22% agreed that HCV treatment should be provided by PCPs, 84% were interested in more HCV training. Willingness to provide treatment was strongly linked to having a high proportion of HCV-infected patients  and availability of other services at the primary care site including HIV treatment , substance abuse treatment  and mental health services .These data suggest that efforts to expand HCV medical provider capacity will be most impactful if they initially focus HCV training on PCPs with a high prevalence of HCV among their patients and existing systems to support HCV care. There are over 3.2 million Americans infected with the hepatitis C virus (HCV) . HCV infPrimary care clinics have traditionally been considered the principal hepatitis C screening venues in the United States. Rates of HCV screening have, however, historically been low. Data from the National Health and Nutrition Examination Survey 2001–2008 (NHANES) reports that only 3.7% of respondents were tested because they or their doctors thought they were at risk for HCV ,5. TheseHistorically, HCV treatment has been provided primarily by hepatology, gastroenterology and infectious disease specialists and not by primary care physicians (PCPs). This has led to significant delays in or failure of HCV care linkage due to a combination of patient and provider level factors including long specialist wait times or lack of willingness by patients to attend HCV care appointments at alternate venues ,9. NewlyAlthough several studies have suggested a need for increased HCV-specific education among PCPs , there aTo update current knowledge and help guide development of interventions to increase access to hepatitis C testing and care services, this study aimed to assess current practices, perceived self-efficacy and willingness to provide HCV care among primary care providers in Maryland.We conducted a cross-sectional survey of primary care providers in care sites within Maryland, USA. The survey was administered via the internet between September 2014 and March 2015.Participants included physicians, nurse practitioners and physician assistant primary care providers (PCPs). PCPs were identified through a listing of providers affiliated with a large academic center  and four large federally qualified health centers  with practice sites throughout Maryland. Each site sent the survey to their internal provider email contact list.A 42 item quantitative online survey hosted by commercial survey software tool, Survey Monkey, was sent by email to participating providers. The survey comprised of multiple choice answer options, with some write-in response options. The survey collected data on provider demographic and clinical site characteristic, knowledge, attitudes and perceptions of substance abuse and HCV care including self-assessments of substance abuse care proficiency, HCV care proficiency and interest in providing HCV treatment. To ensure confidentiality, participants were consented online and assigned unique ID numbers. The survey was intended to be 15 minutes in length and providers could elect to receive a $10 gift card for their participation. The study was approved by the Johns Hopkins University School of Medicine Institutional Review Board.Descriptive analyses were used to describe baseline provider demographic and clinical site characteristics, HCV screening and treatment practices, HCV care proficiency and interest in HCV treatment. A composite score was created for PCP perceived self-efficacy based on five questions on aspects of HCV care for a maximum score of 20. PCPs with a score of 15 or greater were classified as having a self-rated proficiency of being skilled in HCV care and those with a score of 10–15 were classified as having a skill level of average among their peers. Assessment of interest in providing HCV care was based on response to the statement “HCV treatment should be provided by PCPs in the all oral HCV DAA era” based on a 5-point Likert scale ranging from strongly disagree to strongly agree. PCPs were considered to be willing to provide HCV treatment if they responded “agree” or “strongly agree” to this statement. We conducted logistic regression analysis to assess key provider and site characteristics associated with HCV treatment. A p-value<0.05 was considered statistically significant.Of 271 PCPs who received an email invitation, 129 (48%) responded. Nine PCPs did not respond to the statement “HCV treatment should be provided by PCPs in the all oral HCV DAA era” and were excluded from subsequent analysis leaving a total of 120 PCPs for analysis. Six PCPs did not respond to questions used to assess HCV care proficiency but were included for other analysis.As summarized in PCPs reported varying HCV screening practices, with 13% of PCPs reporting rarely screening for HCV, 27% screening in response to clinical factors such as elevated transaminases, 35% screening patients who have HCV risk factors and 25% screening everyone. A majority (78%) of PCPs were aware of the CDC defined “birth cohort” based HCV screening recommendations. These recommendations had been implemented in 60% of these PCPs practice settings with use of an EMR based reminder for HCV screening in 57% of practice settings. A majority of PCPs (89%) reported referring HCV infected patients in their practice for HCV treatment elsewhere with only 56% of PCPs consistently referring (>75%) of their HCV infected patients for treatment. Only 12 PCPs (10%) had treated at least 1 patient for HCV in the preceding year.vs. NP/PA), age, number of years in practice, proportion of patient who were PWID, patient volume or practice site (urban vs. non-urban). In contrast, PCPs reported substantially higher alcohol use assessment and care proficiency with 47% of PCPs considering themselves very knowledgeable or expert.Based on responses to questions on self-rated proficiency in HCV care , the majWhen presented with a clinical case of a patient presenting with a new positive hepatitis C antibody result, 102/120 (85%) appropriately selected HCV RNA testing as the next step in management, however, only 58/120 (44%) correctly identified a need for alcohol use counseling and referral for alcohol use treatment if indicated. The majority 92/120 (77%) of PCPs were aware of the availability of new oral direct acting agents which can cure hepatitis C in 12 weeks or less and most 80/120 (67%) correctly identified HCV genotype as an important part of the HCV treatment decision.One-third of PCPs reported having no prior hepatitis C care training or experience. Among the 71 that reported prior hepatitis C training, this training was received through on-line modules or tutorials (49%), sessions at conference meetings (46%), local CME meeting (37%) and preceptor-ships from another provider (21%). When provided a menu of options through which HCV training could be provided, most (84%) of PCPs indicated one of more avenues through which they would like to receive additional HCV training. Among the 109 PCPs indicating a preference for additional HCV training, 74% noted a preference for online modules, 57%, sessions at conferences, 48% local CME workshops, 26% preceptor-ships with an experienced hepatitis C provider in clinic, and 30% continued discussion with providers treating hepatitis C in their communities.Only 22% of PCPs reported agreement with the statement that PCPs should provide HCV treatment in the all oral DAA era. PCPs who had provided care to a higher number (>100 compared to <100 or >1000 compared to <100) patients in the preceding year were significantly less likely to agree with HCV care provision by PCP. Agreement with PCP provision of HCV treatment was significantly associated with having a high proportion of HCV-infected patients  and availability of other services at the primary care site including HIV treatment (OR 6.5 (2.5–16.5)), substance abuse treatment (OR 3.3 (1.3–8.4)) and mental health services (OR 4.9 (2.0–12.1)) .In this sample of primary care providers in a region with high HCV prevalence ,17–19, aPCPs have been widely considered critical to expanding access to HCV treatment for the large number of individuals infected with HCV in the US ,10. AlthInterestingly, PCPs who either had a large proportion of their patients infected with hepatitis C or had access to support services for populations disproportionately affected by hepatitis C, such as those with HIV infection and substance abuse, were more likely to be willing to provide hepatitis C treatment. While we did not specifically ask why PCPs were willing or not willing to provide treatment, this may reflect either that these providers perceive HCV to be a higher priority among their patients or have a level of experience and comfort with this patient population which makes taking on treatment of a disease that disproportionately affects these populations less of a burden.It was encouraging that the majority of PCPs in our cohort were aware of CDC defined “birth cohort” based hepatitis C screening recommendations and were screening in their clinical practice. As expected, few PCPs were treating HCV. As such, it is not surprising that self-rated HCV care proficiency was, on average low among providers in this cohort. Given the high rates of alcohol use comorbidity in HCV-infected individuals and the negative impact of alcohol use on liver related health in HCV-infected individuals ,21, it iIt is interesting to note that despite low willingness of PCPs to provide HCV treatment, there was high interest in additional HCV training. Interest in additional HCV training was not associated with perceived self-efficacy, suggesting that HCV knowledge is a general area of interest for PCPs. PCPs were also willing to gain this knowledge through a number of avenues including through on-line modules such as those provided by the University of Washington , sessionOur study is limited by the small sample size and the localization of providers to one urban region of the United States. The findings may not be generalizable to rural regions where increasingly HCV infection is found among younger persons who inject drugs. In addition, we were unable to assess bias in our convenience sample of respondents due to lack of information about individuals who refused participation. Our response rates are in line with other provider surveys and suggest validity of our findings.Another limitation of this study is the use of a questionnaire. While questionnaires can be effective in measuring behaviors, attitudes, preferences, and opinions, they may create limitations. In this instance, the fixed-choice questionnaire assumed an understood general knowledge, obliging the respondents to answer questions through a narrow scope.To the extent that provider attitudes predict their receptivity to changes in clinical practice, these data suggest that the largest HCV treatment impact would be achieved by training care providers at sites with many HCV patients and by concurrently creating clinical support systems and time to facilitate the additional effort.


In a recent issue of JAMA, we read with interest the results of The Pregnancy Outcomes Study in Euthyroid Women with Thyroid Autoimmunity after Levothyroxine (POSTAL) study . Thyroidn = 93), where LT4, acetyl-salicylic acid, and prednisolone were given in a non-randomized design, showed a similar miscarriage rate between euthyroid women with thyroid autoimmunity who received LT4 (27.3%) and those who did not (25%) (n = 72) showed that LT4 treatment for thyroid Ab-positive women with normal thyroid function undergoing assisted reproductive technology (ART) did not improve outcomes (miscarriage rate 33% treated vs 52% untreated)  . Both stin vitro fertilization and embryo transfer (IVF-ET) could decrease the miscarriage and improve the live-birth rates among euthyroid women with positive TPOAb (The POSTAL study sought to assess whether LT4 treatment initiated before ve TPOAb . ParticiBefore generalizing these results, however, it is important to have in mind some of the limitations of this well-conducted study. The incidence of miscarriage in clinically recognized pregnancies up to 20 gestational weeks is 8–20%; for women with thyroid autoimmunity, the miscarriage rate can be up to 30% . Taking In conclusion, the POSTAL study showed that LT4 treatment did not benefit euthyroid women with positive TPOAb undergoing IVF-ET. This study adds important knowledge to the field, as LT4 therapy was started early in pregnancy allowing evaluation of early pregnancy outcomes. However, we still need more data to identify subgroups of women with normal thyroid function but positive thyroid antibodies who could potentially benefit from LT4 treatment. Clinicians should share the best available evidence regarding the risks/benefits of LT4 treatment with each pregnant woman enacting a patient-centered approach that takes into account their informed preferences when deciding if LT4 therapy should be started .SM wrote the first draft of the manuscript. NSO and DOK wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Despite intense investigation, no neuroprotective agents for TBI have yet translated to the clinic. Recent efforts have focused on identifying potential therapeutic targets that underlie the secondary TBI pathology that evolves minutes to years following the initial injury. Oxidative stress is a key player in this complex cascade of secondary injury mechanisms and prominently contributes to neurodegeneration and neuroinflammation. NADPH oxidase (NOX) is a family of enzymes whose unique function is to produce reactive oxygen species (ROS). Human post-mortem and animal studies have identified elevated NOX2 and NOX4 levels in the injured brain, suggesting that these two NOXs are involved in the pathogenesis of TBI. In support of this, NOX2 and NOX4 deletion studies have collectively revealed that targeting NOX enzymes can reduce oxidative stress, attenuate neuroinflammation, promote neuronal survival, and improve functional outcomes following TBI. In addition, NOX inhibitor studies have confirmed these findings and demonstrated an extended critical window of efficacious TBI treatment. Finally, the translational potential, caveats, and future directions of the field are highlighted and discussed throughout the review. For instance, many studies have shown that oxidative stress plays a key role in the development of cerebral edema, inflammation, and the secondary neuronal damage found post-TBI 1.22, producing superoxide Initially discovered and characterized as the ROS-generating enzyme in phagocytes responsible for the “respiratory burst,” NOX enzymes consume oxygen to generate superoxide and hydrogen peroxide that can go on to produce other forms of ROS, such as hydroxyl and peroxynitrite 22.1phox (a subunit of NOX 1–4 isoforms) Several groups have examined the role of NOX isoforms in human TBI pathology, and the clinical and post-mortem human data support NOX involvement in TBI. In humans, TBI increases the expression of NOX2 in circulating monocytes 1 day post-injury (dpi), suggesting that TBI can induce systemic inflammatory responses 2.2Animal studies have extensively evaluated NOX elevation and associated pathology following TBI across rodent species and various models of focal and diffuse brain injury. Increases in NOX expression post-injury, which is exacerbated in aged mice phox, p67phox, and p40phox in the injured brain as early as 6 h post-injury phox expression is found in Iba1+ microglia near the lesion Our laboratory and others demonstrated that NOX activity and NOX2 expression in the cortex and hippocampal CA1 region increases rapidly following controlled cortical impact (CCI) with an early peak at 1 h post-injury and a secondary peak from 1 to 4 dpi -/- mice after TBI had a significantly attenuated neurotoxicity on surrounding neurons -/- mice had a profoundly attenuated expression of TNF-α and IL-1β Interestingly, a series of recent studies demonstrated that microglia shift toward a pro-inflammatory state (M1-like) that express high levels of NOX2 at 7 dpi, which is associated with increased cortical and hippocampal neurodegeneration 2.3Although the majority of TBI studies have focused on NOX2, other isoforms have also been examined, and their expression has been characterized in the post-TBI brain phox expression correlated with increased superoxide production Reports of NOX4's involvement in TBI have also emerged recently. NOX4 has been characterized in neurons, astrocytes, and microglia following CCI 3-/- mice (gp91-/- mice) in brain injury employed a surgically induced brain injury (SBI) model -/- mice showed reduced contusion area and apoptosis following TBI that was coupled with a reduction in superoxide and peroxynitrite metabolites in the injured cortex -/- mice have significantly reduced NLRP3 inflammasome expression and activation in the injured cortex following TBI -/- mice may be explained by a correlated decrease of TBI-induced thioredoxin-interacting protein (TXNIP), which has been implicated to directly link oxidative stress to NLRP3 inflammasome activation Perhaps the most definitive evidence we have that implicates a causative role of NOX isoforms in TBI pathology comes from studies utilizing NOX knockout mice. The first report utilizing NOX2hi cells) to the injury site -/- mice, which may be a result of enhanced IL-10 and STAT3 signaling -/- injured mice had attenuated neurotoxicity in cultured neurons Interestingly, deletion of NOX2 also reduced expression of M1-like markers in microglia/macrophages (MG/MP) in the injured cerebral cortex without altering total number of infiltrating macrophages  is a synthetic, small peptide inhibitor that was designed to penetrate cells and prevent the assembly of the active NOX2 complex by inhibiting the association of p475.2-/- mice are frequently used to model chronic granulomatous disease, an inherited disorder characterized by recurrent infections due to defects in innate immunity -/- mice are generally healthy and lack gross phenotypes -/- mice -/- mice.The majority of studies presented in this review focus on the role of NOX2 in mediating neuroinflammation and oxidative stress after TBI, with few recent studies implicating a role for NOX1 and NOX4. Although NOX2 seems the obvious choice for translational pursuit, targeting NOX4 may be advantageous if specific inhibitors are developed. Hospitalized TBI patients have increased susceptibility to potentially fatal nosocomial infections NCT02010242, NCT03226067). Based on these observations, preclinical studies to evaluate the efficacy of GKT137831 in treatment of TBI appear to be needed, as they would help determine the therapeutic potential of targeting the NOX1 and NOX4 isoforms in TBI pathology.While there is no NOX4-specific inhibitor, a new NOX1/NOX4 dual inhibitor GKT137831 has been developed, which may be worth investigating in TBI. GKT137831 has already been shown to reduce neuronal death and degeneration in a rat model of subarachnoid hemorrhage 5.3An important caveat in experimental animal studies on TBI is that the vast majority of animal TBI studies have been conducted in adult male mice of approximately 3 months of age. The lack of age range in the studies is a concern and potentially limiting in translating these basic findings therapeutically to humans. For instance, the highest rates of TBIs are amongst the oldest or youngest age groups who are particularly prone to falling In addition, most animal studies have focused on acute time frames after TBI (hours to days), and there is a paucity of chronic long-term studies after TBI (e.g. months to years after TBI). This is important, because activated MG/MP are present up to 18 years following a single TBI in the human brain 6Despite an abundance of successful pre-clinical studies, the search for an acute neuroprotective drug has yet to deliver promising treatments for TBI. These failures stress the need for innovative therapeutic targets. Though there is significant evidence that NOX enzymes are involved in TBI pathology, many challenges still exist in this pre-clinical phase. Growing the supporting evidence for NOX inhibition across different species, genders, ages, assessment time-points, drug doses, injury sites, and injury models while remaining cognizant of negative data will aid the evaluation of NOX as a target for TBI.


We analyzed GPVI-activated platelets from ST-elevation myocardial infarction (STEMI) patients and matched stable coronary artery disease (SCAD) controls in order to provide novel clues on the degree of involvement of GPVI signaling in the acute event. Firstly, platelets were isolated from systemic venous blood and activated with the GPVI specific agonist CRP (collagen-related peptide). STEMI and SCAD samples were compared by a phosphoproteomics approach. Validations were by immunoblotting in systemic and intracoronary blood from independent cohorts of patients. Twenty-six differentially regulated proteins were identified when comparing CRP-activated systemic platelets from STEMI and SCAD patients, 4 of which were selected for validation studies: PLCɣ2, G6f, SLP-76, and Dok-2. Immunoblot analyses showed these four proteins had higher tyrosine phosphorylation levels in response to CRP in platelets from STEMI patients, being these levels more pronounced at the culprit site of coronary artery occlusion. Moreover, platelet aggregation studies showed a higher response to GPVI agonists in STEMI patients compared to SCAD controls. In conclusion, we show an altered activation state of GPVI signaling in STEMI patients, confirming this receptor as a promising anti-thrombotic target for myocardial infarction.The platelet-specific collagen receptor glycoprotein VI (GPVI) is critical for the formation of arterial thrombosis  ST-elevation myocardial infarction (STEMI) is a particular type of acute coronary syndrome (ACS) that involves intracoronary artery occlusion by a growing thrombus. Despite great progress done in the last decades in the treatment and follow up of patients with STEMI, the incidence of death is still high, being percutaneous coronary intervention (PCI) the most effective method to limit infarct sizein vivo378It is well known that platelets have an imperative role in the development of acute coronary syndromes (ACS) since they attach to the vessel wall and initiate occlusion of the coronary vessel leading to myocardial ischemia, reason why they are considered as key players in myocardial infarctioncollagen-related peptide (CRP). An initial proteomic approach led to the identification of a panel of GPVI signaling biomarkers hyperphosphorylated in STEMI that were validated by immunoblot on larger cohorts of patients. A complementary platelet aggregation-based validation approach was also undertaken. Moreover, GPVI signaling activation was also studied in platelets from intracoronary culprit-site arterial blood taken from STEMI patients during PCI compared to platelets from peripheral blood taken from the radial artery of the same patients. The latter was to test if platelets from blood in direct contact with the thrombus at the occluded coronary artery could reflect better the GPVI signaling activation levels compared to peripheral platelets; this would provide further clues on the relevance of this receptor in the pathological events associated with an acute myocardial infarction.In the present study, we aimed to check the activation state of GPVI signaling in STEMI patients by combining a tyrosine phosphoproteomics-based approach and immunoblotting-based validation assays. Platelets from STEMI patients and matched stable coronary artery disease (SCAD) controls were activated with the GPVI specific agonist For the venous blood studies, platelets from STEMI patients were compared to a control group of age- and gender-matched patients with SCAD. The matching was also based on treatments previous to blood extraction, paying special attention to anti-platelet treatment and statins. Venous and arterial blood samples were collected and platelets isolated as indicated in the Methods section. Besides the expected difference in having a previous history of CAD, there were no other significant differences between both groups, as shown in Our initial goal was to analyze GPVI signaling activation by proteomics in venous blood platelets from STEMI patients in order to provide novel clues on the degree of involvement of this signaling cascade in the acute event. We decided to carry out a small scale proteomic analysis to choose a panel of GPVI activation biomarkers that could be used in larger cohorts of patients. To follow changes in GPVI activation levels only, platelets were activated with the GPVI specific agonist CRP under non aggregating conditions in the presence of inhibitors of secondary mediators. Since GPVI signaling goes primary through tyrosine phosphorylations, proteome analysis was based on immunoprecipitations using the agarose-conjugated 4G10 antiphosphotyrosine antibody, following the same strategy that was previously used to study the GPVI and CLEC-2 signaling cascades11Mass spectrometric analysis of proteins present in the excised bands led to de identification of 26 unique proteins, including several well-known GPVI signaling proteins . Those iIn parallel to the proteomic study, we carried out a small scale analysis of platelet aggregation following activation with CRP and collagen at different doses. Thrombin was used as a positive control. For this analysis, platelets were obtained from an independent cohort of 5 STEMI patients and 5 matched SCAD controls . In agreFrom the differentially regulated proteins identified by proteomics, we decided to choose a panel of four identified in bands showing more differences between groups and of clearly different molecular weight. The latter allowed us to maximize the limited amount of sample available because we could validate the proteins easier by immunoblotting after immunoprecipitations with the 4G10 antiphosphotyrosine antibody. The four proteins selected were (from higher to lower molecular weight): PLCɣ2, SLP-76, Dok-2, and G6f. We then validated those proteins in systemic venous blood platelets from 14 STEMI patients and 11 SCAD matched controls . To follTo gain insight into the disease, we extended the initial venous blood study recruiting an additional cohort of 16 STEMI patients to check GPVI signaling activation in intracoronary arterial blood platelets by immunoblotting . This isThe principal findings of the present study are: (i) proteomic and biochemical identification of a panel of tyrosine-phosphorylated signaling proteins reflecting altered GPVI activation levels in STEMI patients compared to SCAD matched controls; (ii) increased aggregation levels of CRP-, and collagen-activated platelets in STEMI patients, which is consistent with the data mentioned in the previous point; (iii) first GPVI signaling analysis of intracoronary and peripheral arterial platelets from STEMI patients, showing increased GPVI signaling activation levels in platelets at the culprit site of coronary artery occlusion.in vivoDual antiplatelet therapy is indicated in patients who have had ST-segment elevation myocardial infarction because it can safely and effectively reduce mortality and major adverse vascular eventsWe have recently analyzed the proteome of platelets from ACS patients15The enzyme Phospholipase C gamma 2 (PLCγ2): key for the production of second messenger molecules diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) in response to platelet collagen activationThe adapter SLP76: interacts with PLCγ2 to positively regulate GPVI signalingThe adapter docking protein 2 (Dok-2): involved in GPVI signalingG6f, a type I transmembrane adapter protein belonging to the immunoglobulin superfamily, previously identified by us and others as being specifically phosphorylated on Tyr-281 in response to GPVI platelet activation, providing a docking site for the adapter Grb2We chose a double approach to see GPVI activation levels in platelets from STEMI patients. Firstly, we studied systemic venous blood, which is the standard approach. A first group of patients was used to do a small-scale phosphoproteomic study that led to the selection of a panel of signaling proteins altered in STEMI that could be used to follow GPVI activation levels in STEMI patients and SCAD controls. We focused on the following four proteins:in-vitro GPVI activated plateletsThe above proteins were studied in venous blood platelets by immunoblotting and we could demonstrate their tyrosine phosphorylation levels were increased in STEMI versus SCAD controls in response to the GPVI specific agonist CRP. Recent reports by Bigalke and collaborators demonstrated that GPVI surface expression is elevated in platelets from ACS patients181921As an additional validation test, we carried out aggregation assays of platelets activated by GPVI agonists  at different doses. Our results demonstrated an enhanced aggregation of platelets in response to both GPVI agonists in STEMI compared to SCAD controls, suggesting a higher activation of GPVI signaling in the former group, in line with our proteomic and immunoblotting data. It is interesting to mention that we recently showed that GPVI is the main receptor responsible for platelet aggregation by collagenTo further explore the involvement of GPVI in platelet activation associated with an STEMI, we analyzed tyrosine phosphorylation levels of the four biomarkers mentioned above in intracoronary arterial blood platelets, taken from the culprit site (i.e. in direct contact with the thrombus at the occluded artery) in comparison with platelets taken from a peripheral artery. There are precedents of intracoronary studies, using a similar approach, that allowed the detection of platelet activation biomarkers in the intracoronary blood. Indeed, we recently showed an up-regulation of signaling proteins in platelets from intracoronary blood at the culprit site - in comparison with peripheral platelets - in STEMI patientsThe present study has some limitations that should be considered. Sample limitation prevented us from doing more experiments in parallel, such as immunoprecipitations directly against the validated proteins, additional mass spec-based phosphomapping approaches, or carrying out complementary FACS analyses to check platelet activation markers. We are currently starting a new study with different subtypes of CAD patients to carry out further aggregations plus quantitative mass spec analyses to bypass limitations of western blot validations. Ideally, STEMI patients should have been with no medication but this was not possible for obvious reasons . To compensate for this factor, the control group consisted of SCAD patients with the same medication than the acute ones, trying to match in the best possible way all other clinical characteristics between groups.In conclusion, by combining several approaches and studying intracoronary and systemic platelets, the present study demonstrates altered GPVI signaling activation levels in response to receptor stimulation in platelets from STEMI patients, confirming GPVI – and its signaling pathway - as promising anti-thrombotic targets for myocardial infarction.® tubes for analysis. All samples were obtained within the first 12 hours following the initiation of the symptoms, after arrival at the emergency department. Most patients were administered aspirin and P2Y12 inhibitors, primarily clopidogrel, before the blood sample was taken  and developed according to the principles outlined in the Declaration of Helsinki. All methods were carried out in accordance with the approved guidelines, and informed consent was obtained from all subjects. Acute patients included in the study were admitted into a tertiary Spanish hospital presenting with STEMI, defined as angina pain of at least 20 minutes (min) duration with elevated cardiac enzymes and ST-segment elevation of at least 0.1 mV in two or more contiguous leads, or presumably new left bundle branch block. Exclusion criteria were inflammatory or neoplastic diseases, coagulation disorders, platelet-associated disorders, other significant heart disease except left ventricular hypertrophy secondary to hypertension, chronic drug therapy , and having suffered from surgical procedures, major traumata, thromboembolic events or revascularization procedures in the previous three months. At the moment of diagnosis, 25 mL of venous blood or 50 mL of arterial blood  were collected from each patient in 3.2% sodium citrate Vacuetteas taken .For the venous study, besides the STEMI group, blood was also collected from a control group consisting of matched SCAD patients suffering from stable chronic ischemic cardiopathy defined by one of the following: 1) presence of stable angina with documented coronary artery disease  history of coronary angioplasty; 3) history of coronary artery by-pass grafting; and, 4) history of acute coronary syndrome. Such a group was decided to be the most adequate control group due to the clinical characteristics and treatments of the STEMI patients. SCAD patients were required not to have a history of acute cardiovascular event within the year before the inclusion in the study.Both STEMI and SCAD venous-blood was collected from cubital vein in plastic citrated tubes using 21-G needles. Intracoronary samples from STEMI patients were taken during normal hemodynamics procedure using a Pronto V3 catheter (Vascular Solutions), which occludes antegrade flow allowing collecting culprit site blood from the distal artery2527The initial venous blood proteomic screening included 5 STEMI patients and 5 SCAD matched controls . Venous 10-Gly-Cys-Hyp-Gly-NH2, was provided by Dr. Richard W. Farndale, from the University of Cambridge (UK), and crosslinked with SPDP (3–2–pyridyldithio propionic acid N-hydroxysuccinimide ester) by Dr. Yotis A. Senis, at the University of Birmingham (UK). Collagen Reagent HORM® Suspension (KRH) was purchased from Takeda Austria GmbH (Austria). Thrombin was purchased from Sigma .The following agonists were used: Collagen-related peptide (CRP), with the sequence Gly-Cys-Hyp-(Gly-Pro-Hyp)8 platelets/mL were used, whereas for immunodetection studies platelets were used at 4 × 108 platelets/mL. Prior to stimulations, 1 mM EGTA, 10 μM indomethacin and 2 U/ml apyrase were added to washed platelets. For activations, 500-μL aliquots of platelets were warmed at 37 °C for 2 min without stirring and 30 sec with constant stirring at 1200 rpm in a Chrono-log aggregometer, before stimulation for 90 sec and constant stirring with CRP (10 μg/mL) or CRP diluent as control.Fresh blood samples were collected from STEMI patients and SCAD controls. Platelets were isolated by an established method in order to limit contamination from other blood cells and plasma proteins8 platelets/mL. No EGTA or inhibitors of secondary mediators (apyrase and indomethacin) were added. Following the resting step, 300-μL aliquots of platelets were warmed at 37 °C for 4 min without stirring and for 1 min with constant stirring at 1200 rpm in a Chrono-log aggregometer, before stimulation for 6 min with Collagen (5 or 10 μg/mL), CRP  or with thrombin .For aggregations, platelets were resuspended in Tyrodes-HEPES at a concentration of 3 × 108/mL, 500 μL) were lysed with lysis buffer as described previouslyet al. with minor modificationsBasal and stimulated platelets  with a reverse phase nanocolumn  from Proxeon. Ionized peptides were analyzed in a Bruker Amazon ETD ion trap and database search was performed with the Mascot 2.3 search tool  screening SwissProt (SwissProt_2012_10.fasta). Further details on the LC-MS/MS protocol and MS protein identification data can be found in 8/mL, 500 μL) were lysed as described beforeBasal and CRP-stimulated platelets , and ImageJ  version 1.47, and statistical analysis was by Mann-Whitney test or Wilcoxon matched-pairs signed rank test comparing groups. All analyses were performed using IBM SPSS Statistics 20 software for Windows  and graphs done with GraphPad Prism 5 .How to cite this article: Vélez, P. et al. Alteration of platelet GPVI signaling in ST-elevation myocardial infarction patients demonstrated by a combination of proteomic, biochemical, and functional approaches. Sci. Rep.6, 39603; doi: 10.1038/srep39603 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


C2V3 env deep sequencing data (Roche 454) were obtained for 8 paired PBMC and CSF specimens and used for phylogenetic and compartmentalization analysis. Median exposure to ART at the time of sampling was 2.6 years (IQR: 2.2–3.7) and did not differ between groups. We observed that early ART was significantly associated with lower molecular diversity of HIV DNA in CSF (p<0.05), and lower IL-6 levels in CSF (p = 0.02), but no difference for GDS, NFL, or HIV DNA detectability compared to late ART. Compartmentalization of HIV DNA populations between CSF and blood was detected in 6 out of 8 participants with available paired HIV DNA sequences (2 from early and 4 from late ART group). Phylogenetic analysis confirmed the presence of monophyletic HIV DNA populations within the CSF in 7 participants, and the same population was repeatedly sampled over a 5 months period in one participant with longitudinal sampling. Such compartmentalized provirus in the CNS needs to be considered for the design of future eradication strategies and might contribute to the neuropathogenesis of HIV.Even when antiretroviral therapy (ART) is started early after infection, HIV DNA might persist in the central nervous system (CNS), possibly contributing to inflammation, brain damage and neurocognitive impairment. Paired blood and cerebrospinal fluid (CSF) were collected from 16 HIV-infected individuals on suppressive ART: 9 participants started ART <4 months of the estimated date of infection (EDI) (“early ART”), and 7 participants started ART >14 months after EDI (“late ART”). For each participant, neurocognitive functioning was measured by Global Deficit Score (GDS). HIV DNA levels were measured in peripheral blood mononuclear cells (PBMCs) and CSF cell pellets by droplet digital (dd)PCR. Soluble markers of inflammation  and neuronal damage (neurofilament light [NFL]) were measured in blood and CSF supernatant by immunoassays. HIV-1 partial  Human Immunodeficiency virus (HIV) enters the central nervous system (CNS) early after infection and provides the basis for the development of neurocognitive impairment and potentially the establishment of latent reservoirs. Early initiation of antiretroviral therapy reduces HIV reservoir size in the periphery, but no previous study has assessed whether this strategy can also affect the HIV reservoir in the CNS. In this study, we prospectively collected and evaluated cerebrospinal fluid (CSF) and peripheral mononuclear blood cells (PBMC) from a cohort of 16 HIV-infected participants on suppressive antiretroviral therapy (ART) who started ART early (<4 months) and late (>14 months) after the timing of HIV infection. We found that early ART initiation was associated with lower molecular diversity of HIV DNA and lower levels of inflammatory markers in CSF in comparison to late ART start. We also found evidence of compartmentalized HIV DNA populations between the CSF and blood in the majority (75%) of the participants with available paired sequences, including two (66%) participants from the early ART group. Such compartmentalized provirus in the CNS will be important for the design of future eradication strategies and could contribute to the neuropathogenesis of HIV. This has been suggested by previous observations reporting differential emergence of drug resistance mutations between CSF and blood during antiretroviral therapy (ART) failure : 32.5–52.5) selected among participants of the San Diego Primary Infection Resource Consortium (SD PIRC). At baseline (pre ART), the median plasma viral load was 176,000 HIV RNA copies/μl . Participants achieved viral suppression after a median of 76 days (IQR: 47–256) ART start and remained undetectable during the entire follow-up . Participants received ART for a median duration of 2.6 years (IQR: 2.2–3.7) and had suppressed levels of HIV RNA in blood plasma (<50 copies/ml) and in the CSF supernatant (at single copy level) at the time of sample collection. Six out of sixteen participants were on a protease inhibitor (PI)-based ART regimen, 6/16 were on a non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen and 4/16 on an Integrase Strand Transfer Inhibitor (INSTI)-based regimen, all in combination with two nucleoside reverse transcriptase inhibitors (NRTI).While we recruited participants with early and late ART initiation according to study design, the exact categorization (<4 months or >14 months) was performed retrospectively to participant enrollment, but a priori to any molecular data generation or interpretation. The “early ART group” (n = 9) started ART within a median of 1.8 months from estimated date of infection (EDI) (IQR: 1.5–3) while the “late ART group” (n = 7) started ART within a median of 17.2 months from EDI (IQR: 14.8–30.9). Detailed demographic and clinical characteristics of the study population are summarized in env gene  in 8 CSF cellular samples (50%) by nested PCR .Overall, this study comprised 16 participants with baseline samples (9 early ART and 7 late ART) and 3 extra time points from 2 participants (both belonging to the late ART group). Among the 16 baseline samples, we detected HIV DNA from 6 CSF cell pellet samples (37.5%) by ddPCR and amplified the HIV partial Of note, only 5 samples (out of the 13 with detectable HIV DNA) had consistent detection of HIV DNA by ddPCR and nested PCR across both aliquots. This inconsistency across aliquots is not surprising because of the low number of infected cells which increases the proportional impact of unequal cell numbers across the two separate aliquots during processing. Also, the dilution of lysates before the ddPCR droplet generation may have significantly reduced the sensitivity of the ddPCR assay.When comparing the two groups, HIV DNA was detected in 5 out of 9 CSF cell pellet samples tested as part of the early ART group and in 5 of 7 in the late ART group, but this difference was not statistically significant ; HIV DNA was detected in all but one (93.8%) of the 16 PBMC samples.env from CSF cell pellets (n = 8) and PBMCs (n = 14) at baseline. For two participants, we also obtained partial env sequences from one additional time-point (T0338 and T0366). Detailed characteristics of the viral sequences are provided in supplementary To further characterize the HIV DNA population, we sequenced partial Overall, participants in the early ART group presented a lower molecular diversity of the CSF HIV DNA population, as compared to the late ART group (2p = 0.28). When evaluating the compartments separately, this association was significant in CSF , but not in blood . Diversity was significantly higher in PBMC than in CSF by 0.8% , regardless of time to ART. We also included five covariates  separately in the model to examine their potential effects on diversity and the association between time to ART and diversity. None of the covariates was significantly associated with diversity  while the association between time to ART start and diversity remained consistently significant .Next, we used a mixed-effects model where baseline viral diversity was predicted by log-transformed time to ART from EDI as a continuous variable to evaluate its association with percentage of diversity . We obseThe average number of input HIV DNA templates from CSF cells into the first round PCR reaction was estimated using the number of HIV DNA and RPP30 copies (based on our ddPCR data). The median HIV DNA copies per million cells among HIV-positive CSF cell samples was 2,701 copies/million cells . The median number of CSF cells for each ddPCR reaction (estimated by RPP30) was 2,340 (IQR: 1120.5–2700 cells). After adjusting for the different volumes (5 μl for ddPCR and 10μl for nested PCR) and the dilution factor, we estimate that the average calculated HIV template input was 22 copies of HIV DNA (range: 4–64) per reaction. It should be noted that these levels are likely an under-estimate, given the inherent dilution with the ddPCR methods, as described in the method section and above.2p = 0.10). Second, to take into account the lack of consistency across aliquots, we compared diversity measures between cases with consistent versus inconsistent detectability across aliquots (assuming that cases with ddPCR+/nested PCR+ will have higher HIV DNA levels compared to cases with ddPCR-/nested PCR+) and we did not find a significant difference . While the ability to detect a significant effect in our sensitivity analysis was surely limited by the small sample size, this analysis suggests that the effect size of our primary predictor  on molecular diversity of partial env was greater than the effect sizes of each covariate, including the number of template HIV DNA copies (η2p = 0.10) and the number of positive aliquots (η2p = 0.04). Finally, to test the consistency of the diversity measures across blood and CSF, we performed a correlation analysis, and found that molecular diversity in CSF pellets was significantly associated with molecular diversity in PBMC , strongly supporting the validity of our conclusion and measurements within the context of all the aforementioned limitations.To further evaluate if the low HIV DNA input for the sequencing reaction influenced our measures of molecular diversity, we performed additional sensitivity analyses based on our baseline model described above. We first assessed the potential impact of HIV DNA copies on diversity measures by including log-transformed HIV DNA levels (measured in blood and CSF when available) into our model; we found no statistical evidence that the number of HIV DNA copies was associated with any bias in molecular diversity  from CSF cell pellets and PBMCs were obtained for 8 participants, 3 from the early ART group and 5 from the late ART group. Two individuals (both from the late ART group) had additional HIV DNA sequences from a second time-point available . One individual had a third time-point (2 months apart). Compartmentalization was assessed using three distinct methods: distance-based FST test with and without collapsed haplotypes and tree-based Slatkin-Maddison (SM) test. Applying our conservative definition , we observed a significant genetic compartmentalization between the HIV DNA populations sampled from CSF cells and PBMCs in 6 of 8 participants, including 2 individuals in the early ART group (T0104 and T0430)  . Of noteenv sequences from CSF cells and PBMCs (Maximum likelihood (ML) phylogenetic trees were created to evaluate the structure of the HIV DNA populations for participants with paired nd PBMCs .Tree topologies revealed the presence of monophyletic HIV DNA populations in CSF for 7 participants , participants from the early ART group had lower levels of interleukin (IL)-6 .2p = 0.16). When evaluated separately, this association was significant in CSF , but not in blood . Again the five covariates were included in the model to control for their potential effects. The CD4/CD8 ratio was significantly negatively correlated with IL-6 levels , while the other four were not correlated . Regardless of the covariate included in the model, the association between time to ART and IL-6 remained consistently significant .We also used the time to ART as a continuous variable to evaluate its association with the levels of the four cytokines. We observed higher IL-6 levels among participants with the longest time to ART start from EDI, collapsed across blood and CSF . Seven participants had a clear monophyletic population of HIV DNA in the CSF. Overall, our results are consistent with previous studies reporting the presence of compartmentalized HIV RNA in CSF of HIV-infected people very early after infection . The det+ T cells circulating in the CSF after crossing the blood brain barrier but this seems less likely, since HIV-infected CD4+ T cells trafficking from the periphery into the CNS should present an equilibrated viral population in comparison to blood, especially in the setting of suppressive ART. Alternatively, unrecognized isolated HIV replication within the CNS during the period before our study visit might be responsible for our observations. Unfortunately, we did not collect longitudinal CSF samples in time points previous our baseline study visits, as part of the study design.Another open question is the cellular source of this genetically distinct HIV DNA isolated from CSF cells. In our study, we were not able to determine the exact cellular source of the HIV DNA due to technical limitations and the nature of the samples. It is possible that this genetically distinct HIV DNA population detected in CSF might be carried by macrophages or T cells into the CSF or that T-cells circulating in CSF could get infected through contact with HIV-infected macrophages residing in the brain tissue in proximity to the brain vessels . AlternaThe novelty of our study derives from the fact that we evaluated the HIV DNA populations from cells circulating in CSF and we demonstrated the presence of compartmentalized monophyletic HIV DNA populations in CSF from HIV-infected participants receiving suppressive ART, including two participants who started ART during primary infection. Both participants with longitudinal sampling showed sustained compartmentalization at all time-points, and the same monophyletic population was repeatedly sampled from CSF over a period of 5 months in one participant.Despite several technical limitations (described below), our findings are important for the design of future eradication strategies and also to improve our understanding of HIV pathogenesis in the CNS. In fact, the presence of compartmentalized HIV populations has been associated with neurocognitive impairment , 41. SevOur study also evaluated the effect of early ART initiation on selected inflammatory biomarkers in CSF and blood. Increased inflammation has been extensively reported in the CNS during HIV and was often associated with neurocognitive impairment –49 even env gene (~400 bp), we found differences in molecular diversity of the HIV DNA populations in CSF between the early and late ART groups.This study has several limitations. First of all, even though we were able to collect the volumes of CSF necessary to recover sufficient cells by the lysis buffer protocol, the detection of HIV DNA from CSF has been challenging due the low number of cells typically present in CSF in the absence of neurological symptoms and when HIV is suppressed. The low number of input cells might increase the potential for error related to sampling bias, could possibly amplify the number of false positive events from the ddPCR assay and could affect our diversity and compartmentalization analysis. To partially evaluate its impact, we performed multiple sensitivity analysis to address a possible bias in our analysis. Although we acknowledge that the small samples size has limited our statistical evaluation, our primary predictor of interest (i.e. the time to ART initiation) appears to have a greater effect on molecular diversity than the assay-related covariates. Further, we significantly elevated the threshold of compartmentalization detection and specifically included computational tests to increase robustness against significant errors in frequency estimation. Template input was particularly low in some  CSF samples, which could negatively impact our capacity to find unique clades within the CSF: assuming we are simply resampling the most common variants, we are more likely to find that CSF sequences fall within better sampled blood variants. In contrast, despite the possible sampling bias in CSF, we were still able to observe monophyletic CSF variants at baseline in several participants. Also, the reproducibility of the phylogenetic trees with similar variants sampled across longitudinal CSF samples for one participant, suggests that our sequences are likely informative and not substantially affected by random error or sequencing bias. Despite this, and the fact that we are analyzing only a partial region of Another limitation of the analysis is the lack of randomization for the timing of ART initiation, which might introduce some unrecognized biases in our study design. For example, people with more symptomatic infection  will start ART earlier and might also be more likely to present compartmentalized HIV populations. The small sample size also limited our statistical power. Even though some comparisons did not reach statistical significance, effect sizes were medium to large in some cases, supporting that the study was underpowered to answer these questions.Another limitation is inherent in all CSF studies: CSF only approximates events in the brain. Despite this, CSF has provided many important insights into brain events in HIV and other diseases . A high Despite these limitations, our data provide a unique perspective by analyzing HIV DNA populations sampled using CSF prospectively collected from a unique cohort of individuals who started ART and with known EDI. Our study supports the idea that initiation of ART during early infection may limit the diversity of HIV populations and inflammation in CNS. Future studies may want to evaluate the CSF HIV DNA populations in bigger cohorts and include longitudinal assessments prior and after initiation of ART to characterize dynamics of the CNS as a HIV reservoir. Moreover, future studies need to assess the CNS replication competent HIV DNA populations. The presence of unique HIV DNA populations within the CSF during ART might be relevant for future eradication strategies.The study was approved by the Institutional Review Board at the University of California. All adult participants (age ≥ 18 years) provided written informed consent. No children were included in this study.Study participants were selected among HIV-infected men who enrolled in the SD PIRC between 2001 and 2012 and were still engaged in follow-up . All SD Participants were divided in early ART versus late ART groups as follow: 9 were included in the early ART group (≤4 months from estimated date of infection [EDI]) and 7 in late ART group (>14 months from EDI).Paired blood and CSF samples were collected from each HIV-infected participant cross-sectionally. A subset of 2 participants provided a second pair of samples  and one participant provided a third pair of samples (2 months thereafter).env nested PCR as described below , the LPs were performed using atraumatic needle by an experienced physician. None of our study participants reported any complication following the CSF collection. From this larger volume, we obtained a CSF cell pellet and split it into two separate aliquots. Cell pellet lysates (containing HIV DNA) were used for ddPCR and for C2V3 CSF supernatant was used to measure levels of selected markers of inflammation and neuronal damage (described below) and to measure HIV RNA by Aptima HIV RNA assay (Hologic), after concentrating 5 ml of supernatant (with single copy sensitivity). The CNS penetration effectiveness (CPE) index for the most recent ART regimen was determined as previously described . For allFor each participant, neurocognitive functioning was assessed using a standardized clinical battery of seven ability areas consistent with Frascati recommendations for neuroAIDS research  and summThe levels of selected markers of monocyte activation (sCD163), general inflammation (IL-6) and (TNF-α) and monocyte trafficking monocyte chemoattractive protein (MCP)-1 as well as brain damage  was used to quantify the levels of sCD163  from blood plasma and CSF, and NFL in CSF . Electrochemiluminescence multiplex assay  was used to quantify the levels of IL-6, TNF-α and MCP-1 in CSF supernatant and blood plasma. All assessments were performed according to the manufacturer’s procedures.pol gene region: HXB2 coordinates 2536–2662) were measured in triplicate by (dd)PCR [Genomic DNA was extracted from 5 million PBMC for each participant  per manufacturer's protocol. Genomic DNA was also extracted from 1 (out of 2) aliquot of cell pellets obtained from 20 mL of CSF  using direct lysis as previously described , 23. Lev (dd)PCR . Briefly (dd)PCR . The detenv C2-V3 (HXB2 coordinates 6928–7344) region from DNA extracted from CSF cellular pellets and PBMC by nested PCR using specific primers [https://github.com/veg/HIV-NGS and the key steps were summarized briefly bellow:We amplified the HIV-1  primers . Sequenc primers –27. The Raw data were filtered by removing sequences of low quality (q-score of less than 15) using the Datamonkey analysis tool  and aligI is the estimate of mean pairwise intra-compartment genetic distance (TN93) [D is its inter-compartment counterpart [7−109) was handled computationally using an efficient implementation of the TN93 distance calculator (github.com/veg/tn93), which achieves a throughput of 107 distances/second on a modern multi-core desktop. Subsequently, to guard against inference of compartmentalization by skewing of allelic frequencies due to PCR amplification and other biases, we recomputed FST by discarding copy number counts for read clusters (i.e. each cluster was counted as having only one sequence), i.e. all haplotypes are assigned a relative weight of 1. Statistical significance of both tests was derived via 1,000 population-structure randomization/permutation test. Finally, we performed a second tree-based Slatkin-Maddison (SM) test for compartmentalization [ST test, (2) sensitivity test FST with collapsed haplotypes and (3) tree- based SM test.Viral compartmentalization was first assessed by the Fst approach defined as e (TN93) , and πD nterpart . Both qulization . ConservViral haplotypes were realigned using MUSCLE , piped t2 (η2p) was provided as a measure of the strength of association. Statistical analyses were performed using the R statistical language ver 3.3 [Statistical differences between groups (early versus late ART initiation) were examined using linear mixed-effects models with individuals included as random intercepts. The time-to ART variable was dichotomized or log transformed, and outcome variables were rank-transformed when appropriate. When residual variance differed by a specific factor in analyzing untransformed outcomes, we allowed heterogeneous variances across levels of that factor. Differences for sparse variables were detected by Fisher exact test. Whenever possible, partial η ver 3.3  and the  ver 3.3 .S1 FigForty milliliters of CSF were collected from HIV-infected people by lumbar puncture and CSF cells were pelleted down. CSF supernatant was stored at -80°C and subsequently used to measure levels of inflammation and neuronal damage as well as HIV RNA using a Single Copy Assay (Hologic). Cells were resuspended in freezing media and divided into two aliquots stored at -150°C. After thawing, cells were washed with PBS and centrifuged to obtain a dry CSF cell pellet. Cell pellets were directly lysed with lysis buffer and DNA was quantified by ddPCR and amplified by C2V3 Nested PCR.(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Mass extinction events are short-lived and characterized by catastrophic biosphere collapse and subsequent reorganization. Their abrupt nature necessitates a similarly short-lived trigger, and large igneous province magmatism is often implicated. However, large igneous provinces are long-lived compared to mass extinctions. Therefore, if large igneous provinces are an effective trigger, a subinterval of magmatism must be responsible for driving deleterious environmental effects. The onset of Earth’s most severe extinction, the end-Permian, coincided with an abrupt change in the emplacement style of the contemporaneous Siberian Traps large igneous province, from dominantly flood lavas to sill intrusions. Here we identify the initial emplacement pulse of laterally extensive sills as the critical deadly interval. Heat from these sills exposed untapped volatile-fertile sediments to contact metamorphism, likely liberating the massive greenhouse gas volumes needed to drive extinction. These observations suggest that large igneous provinces characterized by sill complexes are more likely to trigger catastrophic global environmental change than their flood basalt- and/or dike-dominated counterparts. Although the mass end-Permian extinction is linked to large igneous provinces, its trigger remains unclear. Here, the authors propose that the abrupt change from flood lavas to sills resulted in the heating of sediments and led to the release of large-scale greenhouse gases to drive the end-Permian extinction. Although other triggers for the end-Permian event have been proposed5, a causal connection between Siberian Traps LIP magmatism and this mass extinction is favored. This causal connection is supported by evidence for a striking temporal coincidence between the two phenomena8, rapid introduction of isotopically light carbon into the marine system9, an abrupt increase in global sea surface temperature (~ 10°C)10, and the physiological selectivity of marine extinction patterns11. These lines of evidence point unequivocally toward a massive, short-lived input of greenhouse gasses  to the atmosphere, which is thought to have been generated in sufficient quantity either by contact metamorphism of crustal sediments during Siberian Traps LIP magma emplacement13 or during LIP plume-related melting at the base of the lithosphere14.Large igneous province (LIP) magmatism1, whereas mass extinction happens on the order of <100 kyr18 , which are often emplaced at different times and over varying intervals, one must critically assess which aliquant of the total magmatic volume, if any, drove biosphere collapse? And further, what trait of this aliquant distinguishes it from the remaining magmatic volume? The responsible volume must be demonstrably emplaced immediately prior to and possibly during the mass extinction, and must have the capacity to trigger massive greenhouse gas release. Recent geochronology on Siberian Traps LIP magmatism and the end-Permian extinction8 has highlighted a distinct temporal association between these two phenomena, but without uniquely identifying the specific extinction-triggering magma volume. This temporal framework nonetheless permits a detailed evaluation of the purported causal connection between magmatism and extinction to determine which subinterval of Siberian Traps LIP magma, if any, induced the mass extinction, and why this particular magma was so deadly.Three issues complicate the proposed causal linkages between mass extinction and LIP magmatism. The first is a significant disparity in the timescales over which LIP magmatism and mass extinction occur; magmatism lasts on the order of 1–5 Myr, with multi-pulsed examples lasting up to 50 MyrHere we identify the deadly subinterval of Siberian Traps LIP magmatism as the initial pulse of sill emplacement into the volatile-fertile Tunguska basin. By considering these results in context with major extinction–LIP couplets within the past 300 Myr, we illustrate the importance of initial widespread LIP sill emplacement into volatile-fertile sediments for driving environmental change on a global scale.8, and the ability to directly compare the relative timing of the two events. Building on the schema presented in ref. 4, we construct a framework to guide identification of the causative LIP subinterval. In this framework, the Siberian Traps magmatic activity can be segmented into three distinct emplacement stages. Stage 1, beginning just prior to 252.24 ± 0.1 Ma, was characterized by initial pyroclastic eruptions followed by lava effusion . Stage 2 began at 251.907 ± 0.067 Ma, and was characterized by cessation of extrusion and the onset of widespread sill-complex formation4. These sills are exposed over a >1.5 × 106 km2 area, and form arguably the most aerially extensive continental sill complex on Earth. Intrusive magmatism continued throughout stage 2 with no apparent hiatus. Stage 2 ended at 251.483 ± 0.088 Ma, when extrusion of lavas resumed after an ~420 ka hiatus, marking the beginning of stage 3. Both extrusive and intrusive magmatism continued during stage 3, which lasted until at least 251.354 ± 0.088 Ma, an age defined by the youngest sill dated in the province4. A maximum date for the end of stage 3 is estimated at 250.2 ± 0.3 Ma6.Recent high-resolution U/Pb geochronology provides a detailed chronology of Siberian Traps magmatism and the end-Permian mass extinction4 shows three notable relationships. (1) Extrusive eruption during stage 1 of Siberian LIP magmatism occurs over the ~300 kyr prior to the onset of mass extinction at 251.941 ± 0.037 Ma8. During this interval, the biosphere and the carbon cycle show little evidence of instability, although high-latitude environmental stress prior to the mass extinction has been observed22  negative δ13C excursion immediately preceding the extinction event4. The remainder of LIP stage 2, which is characterized by continued sill emplacement, coincides with broadly declining δ13C values following the mass extinction. (3) Stage 3 in the LIP begins at the inflection point in δ13C composition, whereupon the reservoir trends positive, toward pre-extinction values.Integration of LIP stages with the record of mass extinction and carbon cycle at the Permian-Triassic Global Stratotype Section and Point (GSSP)23, suggests that only a restricted subinterval of LIP magmatism triggered the mass extinction. Siberian Traps lava eruptions appear to have ceased immediately prior to the extinction, and these lavas likely lack the dissolved CO2 contents required to drive a significant global heating event24, both of which suggest the causative aliquot was probably not extrusive. Initial impingement of a plume head on the lithosphere, and related melting, is also hypothesized to generate significantly higher volatile yields at depth than those released during accompanying LIP lava eruptions14. However, our preferred extinction age model4 indicates that the end-Permian event did not occur during this period of theoretically higher plume-related devolatization (inferred to have initiated at 252.8 Ma14), but postdated it by ~900 ka.The disparity in duration between Siberian LIP magmatism and the end-Permian mass extinction, and the observation that pre-extinction eruption of an estimated 2/3 of LIP lavas resulted in limited deleterious global forcing on the biosphere12. Heating of sediments over the large area encompassed by the sill complex (>1.5 × 106 km2) likely liberated massive volumes of greenhouse gasses25. However, sills were intruded over an interval of ~500 kyr4, while the extinction interval and carbon isotope anomaly are both an order of magnitude shorter. This disparity necessitates that a currently unidentified subinterval of intrusive magmatism during stage 2 is the extinction-triggering aliquant. The oldest sills dated in the magmatic province mark the onset of stage 24, and are the only sills whose emplacement timing is coincident with both the carbon isotope excursion and the onset of mass extinction; all other dated sills postdate these events  are also attributed to rotation of the least compressive stress direction due to high magma flux into the feeder network28, and contrasting mechanical properties between layers of varying density and rigidity29. Once the Siberian sill complex was established, sill intrusion continued into stage 3, coincident with renewed lava effusion. The impetus for stage 3 extrusive magmatism in the northern region of the LIP is unclear, but may be the result of a new eruption center30, or eclipsing of a critical magmatic overpressure threshold at depth26.The exact cause for the observed transition from lava eruptions to widespread sill complex formation during stage 2 is challenging to pinpoint. However, emplacement of these sills occurred immediately after cessation of stage 1 lava extrusion, possibly in response to or as a result of construction of a thick volcanic load atop the lithosphere during stage 1 magmatism Fig. . Numeric30. Consequently, only a small volume of basin sediments experienced contact metamorphism during stage 1 and thus environmental impacts were likely relatively negligible. Not until the Siberian LIP emplacement style changed at the onset of stage 2 from being dominantly extrusive to intrusive was the “volatile-fertile” Tunguska basin subjected to widespread contact metamorphism, thereby maximizing the likelihood of heat transfer and massive volatile yield  dikes in northern parts of the province13C interval at the GSSP, and the sustained elevation in global sea surface temperature10. The biosphere’s ability to buffer itself against prolonged, rather than initially large and abrupt greenhouse gas input, however, likely mitigated many of the deleterious effects of this gas generation24. Sluggish biotic recovery following extinction may well be attributed to stages 2 and 3 magmatism, but the rapid loss in global biodiversity characterizing the mass extinction suggests an equally rapid trigger. Initial intrusion of the Siberian Traps sill complex represents such a trigger, as it was short lived, coincident with the mass extinction, and capable of producing the large amount of climate-altering gasses required to drive biosphere collapse.Lateral sill-complex emplacement continued, subsequent to the initial pulse, but into a basin depleted of its initially high volatile content. Heat transfer to sediments from these later sills still generated greenhouse gasses; their effects are evident from the prolonged isotopically negative δ32, as a sill network built within a volatile-poor substrate will not result in volatile generation on the scale necessary to drive extinction. Some of the largest LIP-related extinction events (>13% genus-level extinction33) in the last 300 Myr are demonstrably associated with widespread sill emplacement into sedimentary basins during Siberian Traps, Central Atlantic magmatic province, and Karoo-Ferrar LIP magmatism17 (Fig. 34. The model presented here suggests a limited role for Deccan magmatism in triggering the K-Pg event, which would shift burden to the contemporaneous Chicxulub impact34. It is plausible that the Deccan LIP and Chicxulub impact shared the causal burden34, and it is therefore possible that neither would have driven extinction of K-Pg magnitude acting alone35. The model presented here suggests that LIPs characterized by a pulse of widespread sill emplacement into a volatile-fertile basin are lethal on a global scale.Our model suggests that LIPs characterized by sill-complex formation are more likely to trigger mass extinction than their flood basalt- and/or dike-dominated counterparts. The composition of sediment into which sills are emplaced is also critically important8. Treatment of this data is available from the corresponding author, S.D.B.Geochronology supporting the model presented here is available through refs. Peer Review File


Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) play a critical role in the generation of vasomotor sympathetic tone and are thought to receive convergent input from neurons at every level of the neuraxis; the factors that determine their ongoing activity remain unresolved. In this study we use a genetically restricted viral tracing strategy to definitively map their spatially diffuse connectome. We infected bulbospinal RVLM neurons with a recombinant rabies variant that drives reporter expression in monosynaptically connected input neurons and mapped their distribution using an MRI-based volumetric atlas and a novel image alignment and visualization tool that efficiently translates the positions of neurons captured in conventional photomicrographs to Cartesian coordinates. We identified prominent inputs from well-established neurohumoral and viscero-sympathetic sensory actuators, medullary autonomic and respiratory subnuclei, and supramedullary autonomic nuclei. The majority of inputs lay within the brainstem (88–94%), and included putative respiratory neurons in the pre-Bötzinger Complex and post-inspiratory complex that are therefore likely to underlie respiratory-sympathetic coupling. We also discovered a substantial and previously unrecognized input from the region immediately ventral to nucleus prepositus hypoglossi. In contrast, RVLM sympathetic premotor neurons were only sparsely innervated by suprapontine structures including the paraventricular nucleus, lateral hypothalamus, periaqueductal gray, and superior colliculus, and we found almost no evidence of direct inputs from the cortex or amygdala. Our approach can be used to quantify, standardize and share complete neuroanatomical datasets, and therefore provides researchers with a platform for presentation, analysis and independent reanalysis of connectomic data. The central tenet of the connectomic approach is that insights regarding both the functional properties of specific neural circuits and general brain organizational principles may be gained by definitively resolving network architecture ] has emerged as a flagship tool for tracing connectomes in experimental animals  in neurons that project to the interomediolateral cell column of the spinal cord (IML), a major site of termination of sympathetic premotor neurons. We then focally microinjected SADΔG(EnvA)-mCherry into the RVLM, selectively restricting its access to TVA-expressing bulbospinal neurons. This enabled us to map brain-wide sources of synaptic input to bulbospinal RVLM neurons using an image alignment tool based on the Waxholm volumetric atlas of the rat brain -mCherry purity was determined by infection of naïve HEK cells and determined to contain approximately 5.3 × 103 unpseudotyped virions per ml. Injection of SADΔG(EnvA)-mCherry in the absence of YTB expression resulted in no labeling in two control experiments.Rabies glycoprotein-transcomplemented SADΔG-mCherry . Control vectors that drive the expression of reporter proteins  were used in initial experiments to determine the time course of protein translation and segment the anatomical boundary of the RVLM. Control vectors were used undiluted and animals sacrificed after 4–5 days.A recombinant herpes simplex type1 (HSV) vector with a retrograde tropism was used to drive expression of rabies glycoprotein, TVA, and YFP (HSV-hCMV-YTB). The gene cassette was derived from the pCAG-YTB plasmid (Addgene 26721) and cloned into recombinant HSV amplicons under the control of the human cytomegalovirus promoter. HSV-hCMV-YTB was supplied at 3 × 10Adult male Sprague Dawley rats (165–500 g) were anesthetized with intraperitoneal ketamine  mixed with medetomidine  and treated with prophylactic antibiotics  and analgesia . Two 500 nl injections of HSV-hCMV-YTB were made over 5–10 min at coordinates corresponding to the left T2 IML as previously described -mCherry by recording antidromic field potentials evoked by stimulation of the facial nerve  neurons outside of the RVLM . Such neurons were easily identified by their dual expression of mCherry and YFP: contaminated experiments were excluded from analysis. Optimal results were obtained when SADΔG(EnvA)-mCherry was injected 24 h after HSV-hCMV-YTB and animals kept for a further 6 days.2HPO4, 26.6 mM NaH2PO4, 876 mM sucrose, 5 mM ethylene glycol) for storage at − 20°C.Animals were euthanized with sodium pentobarbital  and immediately transcardially perfused with 300 ml ice cold heparinized saline followed by 300 ml 4% paraformaldehyde. The brain and thoracic spinal cord were then removed and post-fixed overnight. With the assistance of a brain matrix, brains were cut coronally 2 mm caudal to the olfactory bulb and mounted frontal pole down on a vibratome plate so that the ventral surface of the brain was approximately perpendicular to the plate. The entire brain was sectioned at 50 μm in the coronal plane using a Leica VT1200S vibrating microtome and collected in 4 bins in 0.01 M Tris-phosphate buffered saline (TPBS). Pot 1 was mounted directly onto glass slides to maintain section order. Brainstem sections from a second pot were processed for YFP and TH immunoreactivity so that starter neurons could be identified. The other pots were transferred to cryoprotectant solution . Images were obtained from sections that were immediately mounted during cutting, preserving order, except for sections from the RVLM region; these data were obtained from alternative sections that were processed for YFP and TH immunoreactivity. Neurons that contained both YFP and mCherry were classified as starter neurons and were further sub-classified as TH-immunoreactive (C1) or TH-negative (non-C1). Input neurons and C1/non-C1 starter neurons were manually annotated on each image using Zen software; the pixel coordinates of annotated neurons were then extracted from file metadata using the ImageJ/FIJI package  and tabulated.Epifluorescence imaging at 10x permitted efficient imaging of whole-brain datasets at the expense of sensitivity, biasing against lightly labeled or small neurons. This was particularly problematic for detection of lightly-labeled TH-immunoreactive starter neurons; confocal reimaging  of three sections containing starter neurons previously imaged and analyzed as described above indicated that approximately a third of the C1 starter neurons detected under confocal had been identified as non-C1 under epifluorescence (19/27 vs. 13/27). This problem did not apply to detection of rabies-infected neurons, which were unambiguously labeled.Image alignment and anchoring was achieved using a beta version of the AligNII tool embedded in Navigator-3 (N3), a web-based data management system currently under development of the University of Oslo Neuroinformatics group , resulting in a 3d model of the Waxholm brain populated with points corresponding to identified neurons. Another script was then used to automatically quantify the number of input neurons that lay within each segmented region . The Imaris-rendered Waxholm brain (containing the RVLM connectomic dataset) is available as a supplementary download and can be used freely by other researchers (http://datadryad.org/resource/doi:10.5061/dryad.q5t5s). The dataset can also be viewed with the free Imaris SceneViewer program (www.bitplane.com). The voxel coordinates of starter and input neurons are included in tabulated format in Data Sheet The raw MRI data and corresponding segmentation model of the Waxholm Sprague Dawley rat were downloaded from the International Neuroinformatics Coordinating Facility Software Center . Contours enclosing pixels that contained >2 neurons/10 pixel radius in the horizontal plane were converted to an image stack, imported into the virtual Waxholm rat brain using Imaris, and surface rendered to define the boundaries of the RVLM , assigned initially at random and iteratively refined as data points are sequentially accumulated within a cluster. Analysis was performed and density plots generated in “R project” . The voxel coordinates of input neurons segregated by cluster are provided in Data Sheet Microinjection of HSV vectors at the thoracic spinal cord drove reporter expression in bulbospinal neurons within 12 h, with maximal expression apparent by 2–4 days. Reporter-labeled neurons spanned the RVLM, rostral ventromedial medulla (RVMM), and midline raphe region, and were identified in other sympathetic premotor nuclei such as the C3, paraventricular, A5, and locus coeruleus (Supplementary Image n = 236) and corresponds well with the pressor region that encompasses the rostral RVLM and perifacial zone . Subsequent injection of SADΔG(EnvA)-mCherry in the RVLM resulted in the primary infection of bulbospinal RVLM neurons. As shown in Figure Data from 4 animals were selected for detailed connectomic analysis. As illustrated in Supplementary Image Neurons were counted on every fourth section: on average 38 starter neurons were identified per animal range: 19–67), of which 40% (30–45) were confirmed as TH-positive C1 neurons under epifluorescence (which underrepresents the proportion of C1 starter neurons by up to 50%: see Methods–Imaging). Seventy eight percent (74–87%) of starter neurons lay within the Waxholm RVLM boundary defined above Figure . Ectopic9–67, of Monosynaptic input neurons were identified throughout the brain as mCherry, non-YFP neurons. 1298 input neurons were identified in total [325 (220–561) per animal], corresponding to an input: starter ratio of 9.8 (4.4–13.4). The overall distribution of input neurons is shown in Figures A script that automatically counts the number of neurons within each region of the Waxholm atlas was used to quantify the regional distribution of input neurons. These data are graphically represented in Figure K-means analysis was used to objectively group input neurons based on their spatial distribution. We empirically determined that 12 was an appropriate number of clusters (k) by which to partition the dataset by plotting the percentage of variance explained by clusters generated for k-values 1–20 and identifying a point at which the increase in explained variance was marginal for further increases in k, denoted by an “elbow” in the curve  and contralateral (Cluster 11) A5 adrenergic cell group, subcoeruleus, Kölliker-Fuse, and medial and lateral parabrachial nuclei. The single midbrain cluster (Cluster 8) incorporated the lateral and ventrolateral periaqueductal gray (PAG) and colliculi, and a hypothalamic cluster (Cluster 12) composed from neurons residing within the paraventricular hypothalamic nucleus, including confirmed vasopressinergic neurons Figures , 6E and The objective of the current study was to definitively and quantitatively map the afferent connectome of putative RVLM sympathetic premotor neurons, a regulatory axis through which convergent sensory and limbic inputs are thought to summate to produce baseline sympathetic nerve activity. We identified prominent inputs from well-established neurohumoral and viscero-sympathetic actuators such as the area postrema and NTS, and inputs that spanned medullary autonomic  and respiratory nuclei , as well as supramedullary autonomic nuclei  Dampney, . HoweverThe minor input received from supramedullary centers is unlikely to represent a limitation of SADΔG(EnvA), as distance is not thought to impair its labeling efficiency  is inexact; it can be estimated by examining the degree of variance accounted for by iterative increases in k and identifying the “elbow” after which the relationship between k and variance becomes linear . However, as this value is a function of the variance of the dataset, one would expect k to increase if more experimental data were added—in other words, if one analyzed 10,000 input neurons instead of 1,000, the sensitivity of the analysis would be higher and it would resolve clusters not currently detectable. Second, cluster analysis was performed on the pooled connectomic dataset, rather than on each individual experiment. Although the overall normalized distribution of inputs was reproducible between experiments, this means that experiments in which a large number of input neurons were detected influence cluster detection more than those in which fewer inputs were detected. Taken together, cluster analysis of connectomic datasets should be seen as a useful platform for breaking complicated datasets down into a readily digestible overview, rather than a definitive structural analysis tool.Like other mesoscale connectomes, the RVLM connectome is diffuse and highly non-uniform in its distribution. This level of complexity makes it difficult to conceptualize as a whole, and makes identification of its major subdivisions susceptible to operator bias. Principle component analysis  provides a simple and unbiased way to do this but suffers some potential drawbacks. First, selection of the appropriate number of clusters  eliminates potential errors caused by imperfect cutting angle or tissue distortion. Second, the compact size (current study <100 kb) and standardization of the positional metadata make it amenable to sharing and independent reanalysis, and eliminates the need for other investigators to directly interact with cumbersome raw imaging data (current study >300 Gb). Routine publication of such metadata would enable researchers to present complex neuroanatomical datasets in a standardized and transparent format that improves utility and reproducibility.The promise of the connectomic approach is reflected by its widespread adoption and the continued refinement of connectome-tracing viral vectors (Osakada et al., Conceptualization: SM, AG, and AA; Methodology: BD, RN, and SM; Software: JB; Formal analysis: SL, BD, AT, and SM; Investigation: BD, AT, SL, CM, and SM; Resources: RR, PB, RN, and JB; Data curation: SL, BD, and SM; Writing—Original Draft: BD, AG, and SM; Writing—Review and Editing: All authors; Visualization: BD and SM; Supervision: SM; Project Administration: SM; Funding Acquisition: SM, AA, and AG.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.