Objective: To evaluate the tolerability and safety of Octagam® 5% and 10% across all indications, ages, and treatment regimens, using data from four non-interventional post-authorization safety studies (PASS); this analysis was performed following changes in the preparation of raw material used to manufacture Octagam. Methods: All four studies included in- and out-patients prescribed Octagam for treatment of their medical condition. Physicians used case report forms to document baseline demographics, Octagam treatment details, and data on the efficacy of Octagam, and recorded all adverse drug reactions (ADRs) and other safety data. Results: Altogether 21,780 infusions of Octagam in 2,397 patients were included in our analysis. The most frequent indication for Octagam was secondary immunodeficiencies , followed by primary immunodeficiencies . During the individual patient observation, 83% of SID and 67% of PID patients were free of any infection. In up to 85% of all investigator assessments, Octagam was rated to have a favorable effect. In autoimmune diseases, investigators assessed Octagam as being beneficial in 70% (immune thrombocytopenia) up to 100% (Guillain-Barré syndrome), depending on the indication. The majority of patients (92%) tolerated Octagam treatment without any ADR. The overall incidence of reported ADRs was 1.0% for all infusions. The majority of ADRs were considered non-serious (93%) and mild or moderate (87%) in severity. No unexpected ADR signal was detected. Conclusions: This analysis demonstrates that the changes in the preparation of raw material used to manufacture Octagam did not affect the safety profile of Octagam® 5% and 10%.  Immunoglobulins are widely used in both, immunodeficient patients, to provide antibodies to prevent viral and bacterial infections (replacement therapy), and in patients with autoimmune and inflammatory diseases, mediating immunomodulation and attenuating inflammatory responses , 5, 6, 7® 5%  is a sterile ready-to-use, sucrose-free liquid preparation of highly purified immunoglobulin for intravenous administration (IVIG), which was originally approved in Europe in 1995 for the treatment of primary (PID) [® 10% was originally approved in Europe in 2008 for the same indications [® 5% and Octagam® 10% are also approved as immunomodulation therapy in Guillain-Barré syndrome (GBS) and Kawasaki disease, chronic inflammatory demyelinating polyneuropathy (CIDP) in selected geographical areas and in allogenic bone marrow transplantation and congenital AIDS. Octagam® 5% and Octagam® 10% were available on the market with an amended manufacturing process. An additional chromatography step was introduced in the preparation of raw material paste used to manufacture Octagam to increase product safety. In order to test the positive risk-benefit ratio of the two Octagam products, a plan for integrated safety analysis was created with the objective to document and evaluate the tolerability and safety of Octagam® 5% and 10% in any indication, age group, or treatment regimen. Data from two ongoing and two newly established non-interventional PASS studies was used to analyze the incidence and the nature of ADRs from at least 20,000 infusions with Octagam® 5% or Octagam® 10%. In June 2011, OctagamData from four non-interventional, post-approval, multicenter studies were collected and included in this integrated safety analysis. The designs of these studies are presented in ® 5% and Octagam® 10% were included if their physicians had prescribed Octagam® (5% or 10%) for their medical condition. Participating physicians decided the length and dosage of Octagam treatment based on disease type and severity as well as the patients’ clinical condition. For all four studies, in- and out-patients receiving OctagamIn all studies, physicians documented the data on case report forms (CRF) in accordance with each of the study protocols; for two studies, paper CRFs were used, while the other two studies used electronic CRFs (eCRFs). Collected information included baseline demographics  and Octagam treatment details . Efficacy in preventing infections in patients with PID or SID was assessed by asking physicians to rate every 3 months the influence of treatment on patients’ disease as either “favorable”, “unchanged”, or “unfavorable” with regard to infection frequency, severity, duration, and antibiotic consumption. In patients with indications other than PID or SID, physicians were asked to rate in the 3-months observation periods how the patients’ underlying diseases had developed, using the outcome categories “improved”, “unchanged”, or “deteriorated”. One study also rated the influence of treatment on the course of the patients’ diseases at the end of the individual observation period as “beneficial”, “unchanged”, or “unfavorable”. In the course of the studies, physicians recorded all adverse drug reactions (ADRs) and other safety data. An ADR was defined as any noxious and unintended response to the observational drug, implying that a causal relationship between the observational drug and an adverse event is at least a reasonable possibility, i.e., a causal relationship cannot be ruled out. Adverse events that were not considered to be related to treatment with Octagam were not reported. ADRs were classified according to the nature of the event (MedDRA preferred term), frequency, severity , and causal relation to Octagam . An independent Data Monitoring Committee (DMC) reviewed all safety relevant information, analyzed the nature of ADRs, and assessed the causality of serious ADRs in addition to the investigator and Octapharma. A definite relationship involved an adverse event that occurred within a reasonable time period from drug intake that made a causal relationship plausible, and that could not be explained by the disease or any other drug, and, for which re-challenge, if necessary, was satisfactory. An unlikely relationship involved an adverse event that occurred with a time from drug intake that made a causal relationship improbable but not impossible, and for which disease or other drugs could provide plausible explanations. In the case of unclear or implausible causality, the DMC made a final decision as to whether there was any likelihood of the adverse event being related to Octagam. The physicians were to report serious ADRs immediately (within 24 hours) to Octapharma. Serious ADRs were defined if at least one of the following criteria was applicable: event was life-threatening, required in-patient hospitalization or extended existing hospitalization, resulted in persistent or significant disability or incapacity, resulted in congenital anomaly/birth defect, or patient died. Non-serious ADRs as well as any other safety information were reported at the time of recognition if possible and no more than 10 working days thereafter. Data from all four studies were entered into a common database. Data on paper CRFs were entered manually or transferred electronically into the database whereas data documented using eCRFs were transferred directly using a web-based electronic data-capture system. Where appropriate, data was stratified according to country and indication, age groups, or patient’s gender. Statistical analyses for this study were performed using the software package SAS release 9.3 . All safety and efficacy variables were analyzed by descriptive statistical analyses and included the absolute and relative frequencies, arithmetic mean, standard deviation (SD), minimum, median, and maximum, and two-sided 95% confidence intervals (CIs), where appropriate. If required by the national regulations, the study protocol was reviewed and approved by each study site’s Independent Ethics Committee or Institutional Review Board before the start of the study. Patients provided written informed consent prior to study entry where required by local regulatory requirements. ® 5%  than with Octagam® 10% (961 patients) and some patients received both product strengths. In total, 2,397 patients, who had collectively received a total of 21,780 infusions of Octagam, were included in this safety analysis. Patients at any age with any indication for Octagam treatment were included . The ageOverall tolerability In total, 210 ADRs were reported in 191 patients during the observation period. The vast majority of ADRs were non-serious (93%) and mild or moderate (87%) in severity . The ove® 5% was 8.7 mL/kg/h (14.5 mg/kg/min) and 7.2 mL/kg/h (12 mg/kg/min) for Octagam® 10%. However, no correlation was found between infusion rates and ADR incidence. This integrated safety analysis showed that in most cases (88%) no premedication was used prior to infusion. Octagam was also well tolerated at high infusion rates. The maximum infusion rate was documented in two of the four studies. The documented maximum infusion rate for OctagamTolerability in patient subgroups Combined data from all four studies revealed some differences by indication in tolerability . The mosOut of 2,397 enrolled patients in four studies, 15 patients experienced at least 1 serious ADR (SADR). 10 SADRs occurred in SID patients, no SADRs were documented among PID and ITP patients. The remaining 5 SADRs occurred in patients with various other indications. The nature of these SADRs may be divided into 3 categories: The first category consists of 8 cases of immediate-type hypersensitivity reactions, which were assessed as related to Octagam by the investigators and by Octapharma. In 6 cases, the patients experienced chills and other symptoms either during the infusion or up to 4 hours after the infusion. Of these, 1 hypersensitivity case was assessed as mild and the patient received fluid replacement therapy; 3 hypersensitivity cases were assessed as moderate and were treated with fluid replacement in 1 case, glucocorticoids in the remaining cases and additional antibiotic therapy in 1 case; 2 cases were assessed as severe and were treated with glucocorticoids. The remaining 2 cases comprised severe headache during the infusion in 1 case and 1 circulatory insufficiency during infusion. However, all 8 patients recovered without sequelae. At least 2 patients continued the Octagam therapy after their event without further problems. The second category consists of 3 SADRs that can be classified as thromboembolic events (TEEs); 1 case each of thromboembolic occlusion of the aorta abdominalis, thromboembolic occlusion of the femoral artery, and pulmonary embolism. The total rate of TEEs per infusion across all four studies was 0.014%  The causal relationship to Octagam treatment was assessed as unlikely in 2 out of 3 cases and as possible in 1 case by the DMC. The third category of the remaining 4 SADRs comprises a heterogeneous group: Rash 28 days after last infusion, classified as unlikely related to Octagam treatment by Octapharma; renal colic 2 hours after last infusion, assessed as unlikely related to Octagam treatment by investigator and Octapharma; gastric ulcer 5 days after last infusion, assessed as unlikely related to Octagam treatment by investigator and Octapharma; the last case includes exacerbation of dyspnea during infusion, assessed as probably related to Octagam treatment by investigator. During the individual patient observation, 67% of PID and 83% of SID patients treated with Octagam were free of any infection. The most common infections concerned the upper respiratory tract in both groups. Physicians made 1,317 assessments for SID patients, with a mean assessment period of 3.4 months. The influence of Octagam on infection frequency was rated as “favorable” in 85% of patients. Furthermore, Octagam was rated to have favorable effects on infection severity, infection duration and the consumption of antibiotics in 83%, 79%, and 79% of patients, respectively. Among 530 assessments in PID patients, with a mean assessment period of 3.6 months, physicians considered “favorable” improvements in the frequency of infection in 80%, the severity of infection in 77%, the duration of infection in 74%, and the consumption of antibiotics in 72% ; for allFor ITP patients, physicians made 125 assessments and rated the clinical appearance as “improved” in 70.4%. In only 0.8%, they rated the clinical appearance as “deteriorated” and the remainder (28.4%) was “unchanged”. In GBS, the influence of Octagam on the disease course was rated at the end of treatment: In all 4 cases, the rating was “beneficial”. In other neurologic autoimmune diseases, physicians made a total of 687 assessments, which revealed that the clinical appearance was stable in 72.3% or even improved in 26.1%. Several review articles have addressed the safety of various IVIG preparations, finding that the most frequently reported adverse events following IVIG treatment are mild infusion-related reactions such as headache, nausea, fever, chills, and myalgia , 11, 12.Importantly, the adverse drug reaction profile observed in this study does not differ from that expected based on previous studies of Octagam and of other IVIG preparations. The ADRs reported in this analysis were similar to those observed in a 10-year prospective observational  study of Octagam in patients with various PID and SID and autoimmune diseases; these studies reported ADRs in 4.2% of all patients and in 0.35% of all infusions, with most ADRs graded as mild or moderate . Finding® , Tegeline® , Immunoglobulin® , Flebogama® , Vigam® (Bio Products Laboratory Limited (BPL), Hertfordshire, UK), Kiovig® , immediate infusion-related adverse reactions occurred in 38 of 1,765 infusions (2.15%), most commonly chills, fever, headache, nausea, malaise, and myalgia [®) reported 67 ADRs in 27 subjects (30.6%), which were mild to moderate in intensity and mostly transient; the most frequent ADRs reported in this study were headache, pyrexia, vomiting, and chills [®  infusions, there were 225 events attributed to Intratect®, resulting in an ADR rate of 1.0% per infusion [Results of the present analysis are consistent with a prospective audit conducted in 459 patients who were established on stable IVIG treatment having received at least six infusions uneventfully; as patients in this study were not selected, but rather the participating centers entered all patients receiving IVIG treatment, this study design is consistent with a non-interventional study. Over a total of 13,508 infusions given, 111 adverse reactions occurred, of which 91 were mild  and 20 were moderate, with an overall adverse reaction rate of 0.8% . Notably myalgia . A recend chills . In a muinfusion . While direct comparisons of the adverse event profiles of the various IVIG formulations are not possible, available data suggest that overall adverse event profiles are broadly similar. Any differences that do occur between formulations are likely explained by differences in manufacturing processes. Such variances may result in differences in the biochemical composition of the final product, including differences in sodium content, sugar content, osmolality, protein content, stabilizing agents and pH , 21, 22.® 5% and 10% are not associated with a high incidence of TEEs and this is consistent with the approximate frequency  . From June 2011 on, an additional and now mandatory chromatography step to remove activated factor XI was integrated in the standard preparation of raw material used to manufacture Octagam. In addition, a Thrombin Generation Assay (TGA) was introduced in routine Octagam batch release (with the purpose to show that FXIa is below a limit defined by regulatory authorities). This safety analysis demonstrated that these new steps decreased the unexpected incidence of TEEs noticed in 2010 to rates observed in the original clinical studies of Octagam and improved the benefit-risk ratio of both Octagam preparations , 9. An aOne of the strengths of this analysis is the fact that when data from multiple studies are pooled, the statistical power of the data can be increased. Additionally, the studies included in this analysis involved patients from several European countries as well as from the US. A further advantage is the inclusion of both Octagam product strengths and the use in various indications. There are some limitations to this safety analysis. First of all, there was a large variety in patient numbers in each of the studies analyzed. While this does not affect the primary analysis, any analysis by age or indication should be interpreted with caution. Furthermore, the fact of substantial variations between studies, due to the different study designs, various indications and different countries involved, must also be taken into account when interpreting the results of this study. ® 5% and 10% investigated in this integrated safety analysis are well tolerated and safe. There were no unexpected safety issues and the ADR profile observed was consistent with that previously reported for Octagam and other IVIG products. These results demonstrate that the change in the manufacturing process has effectively re-established the positive risk-benefit ratio of the two Octagam products. In conclusion, Octagam was found to be safe and well tolerated over a broad group of patients and indications under conditions of routine medical use. This analysis demonstrated that the preparations of OctagamThe studies were funded by Octapharma. The authors were employees of Octapharma USA Inc., Hoboken, NJ, USA, Octapharma GmbH, Langenfeld, Germany or Octapharma Pharmazeutika Produktionsges.m.b.H., Vienna, Austria at the time of study realization.


CT) can be used as a novel noninvasive method for diagnosis of ischemia-causing coronary stenosis.The instantaneous wave-free ratio (iFR) closely related to fractional flow reserve (FFR) is a adenosine-independent physiologic index of coronary stenosis severity. We sought to evaluate whether iFR derived from coronary computed tomographic angiography  curve was used to select the cut-off value of iFRCT for diagnosis of ischemia-causing stenosis. The diagnostic performances of iFRCT, coronary CTA, and iFRCT plus CTA for ischemia-causing stenosis were compared with ROC curve and Delong method.We retrospectively enrolled 33 patients (47 lesions) with coronary artery disease (CAD) and examined with coronary computed tomographic angiography (CTA), invasive coronary angiography (ICA), and FFR. Patient-specific anatomical model of the coronary artery was built by original resting end-diastolic CTA images. Based on the model and computational fluid dynamics, individual boundary conditions were set to calculate iFRCT and FFR had linear correlation . ROC analysis identified an optimal iFRCT cut-off value of 0.82 for categorization based on an FFR cut-off value 0.8, and the diagnostic accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of iFRCT were 78.72%,70.59%, 83.33%,70.59%, and 83.33%, respectively. Compared with obstructive CAD diagnosed by coronary CTA (AUC = 0.60), iFRCT yielded diagnostic improvement over stenosis assessment with AUC increasing from 0.6 by CTA to 0.87 (P < 0.01) and 0.90 (P < 0.01) when iFRCT plus CTA.On a per-vessel basis, iFRCT is a promising index improving diagnostic performance over coronary CTA for detection of ischemia-causing coronary stenosis.In conclusion, iFR Invasively measured fractional flow reserve (FFR) is widely recognized as the gold standard in evaluating the functional changes of coronary stenosis.–10 Revascularization therapy based on FFR is a reliable method for improving prognosis and reducing medical costs, and it has been included in the guidelines as the gold standard for qualitative diagnosis of obstructive CHD and treatment strategy. However, in practice, the application of FFR is limited due to its invasive nature, and some patients find it hard to accept vasodilatory drugs and the high cost.,12In recent years, coronary computed tomography angiography (CTA) has been proved to be a favorable means of morphological examination for coronary artery stenosis. The ADVISE study reported that iFR had a good correlation with FFR  and showed excellent diagnostic efficiency. When FFR < 0.8, the area under the receiver operating characteristic curve (AUC) of iFR amounts to 0.93. The JUSTIFY-CFR study showed that iFR had a better correlation with coronary flow velocity reserve (CFVR) than FFR, which could be used in functional tests of vascular lesions independent of FFR. Also, van de Hoef et al investigated iFR and FFR against a combined reference of myocardial perfusion scintigraphy and hyperemic stenosis resistance (HSR), and they found that among 19% of the cases where iFR disagrees with FFR, FFR did not always produce highly accurate results, indicating that FFR is not necessarily a better discriminator of coronary ischemia than iFR. Moreover, iFR avoids the administration of vasodilatory drugs needed for FFR, and it has less variability and fewer repeated measurement differences compared with FFR (−0.0005 ± 0.002 vs 0.01 ± 0.04).Recently, a new derivative pressure indicator (instantaneous wave-free ratio (iFR)) in the resting state has been introduced, which can be measured without administration of vasodilatory drugs. iFR is defined as the ratio between the mean instant pressure distal to coronary stenosis and the mean pressure at the aortic root during wave-free period. The internal resistance of coronary artery is the lowest and most constant during wave-free period (defined as the diastolic window), which ranges from 25% of the way into diastole to 5 ms before its ending (close to 0). This is similar to the internal resistance of coronary artery achieved by vasodilator drugs during direct measurement of FFR.CT) has been used for integrated evaluation of anatomical and physiological functions of coronary stenosis, and it showed that the detection of hemodynamically significant stenosis evaluated by FFRCT correlated well with invasive FFR.–19 However, the application of iFR derived from coronary CTA (iFRCT) for hemodynamic assessment of coronary stenosis has not yet been studied. Therefore, the purpose of our study was to evaluate the correlation between iFRCT and FFR, explore the diagnostic efficacy of iFRCT in the diagnosis of functional ischemia, and investigate the clinical value of iFRCT combined with CTA in the evaluation of coronary stenosis.So far, FFR derived from coronary CTA  were retrospectively collected. Inclusion criteria: patients who underwent coronary CTA, invasive coronary angiography (ICA), and FFR in a nonemergent setting. Exclusion criteria: time between procedures exceeding 2 months, major interprocedural adverse cardiac events , significant decrease of left ventricular function, complicated congenital heart diseases, previous coronary artery bypass surgery or stenting, installed pacemaker, artificial heart valves, bifurcation stenosis, chronic total occlusion, nondiagnostic quality of CTA data, and body mass index (BMI)≥35. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. The study was approved by the ethics committee of Shengjing Hospital of China Medical University, the ethics committee of the First Affiliated Hospital of China Medical University, the ethics committee of General Hospital of Shenyang Military Command, the ethics committee of Dalian Central Hospital, and the ethics committee of General Hospital of Guangdong Province. Informed consent from patients was not needed because of the retrospective nature of this study.2.2,21 CTA was performed with ≥64-slice multidetector scanners . Patients with heart rate >70 bpm were treated with (β)-blockers, and no nitroglycerin was given to any patient before CT examination. During the scanning, 60–80 Ml contrast agent  was administrated through intravenous injection (4–5 mL/s), followed by rinsing with 20 to 30 mL saline. Retrospective electrocardiography (ECG)-gated spiral scanning or prospective ECG-triggering axis scanning was used. Scanning parameters included: collimator width of (2 × 64)/128/320 × 0.5/0.625 mm; 100 or 120 kV; effective tube current of 400 to 700 mA. Effective dose (ED) for CTA was calculated using the following formula: ED (mSv) = (dose length product) × 0.014. The effective radiation dose was 1.5–10.8 mSv.All the CTA scanning was carried out in accordance with the guidelines recommended by the Society of Cardiovascular Computed Tomography.The original data were reconstructed using standard convolution function, with a sharp convolution function reconstruction algorithm used for cases with severe coronary artery calcification. The best quality end-diastolic phase images were selected for further analysis. Images with quality scores of 2 to 4 points were included in the analysis. The evaluation of stenosis was carried out using blood vessels as the unit, which consisted of the left anterior descending artery , left circumflex artery (including the middle branch), and right coronary artery (including the right posterior and posterior descending branches). Stenosis was classified as 4 grades through visual inspection: mild stenosis (lumen diameter reduced by <50%), moderate stenosis (lumen diameter reduced by 50%–69%), severe stenosis (lumen diameter reduced by 70%–98%), and subtotal-to-total occlusion (lumen diameter reduced by 99%–100%). Per-vessel stenosis severity was evaluated by the extent of the maximal stenosis (lesion of interest) within the vessel.Radiologists with 7 years of experience or more evaluated image quality and analyzed coronary stenosis. The image quality of coronary CTA was assessed using a 4-point Likert scale .2 located on the vascular wall or in the surrounding lumen, which could be clearly distinguished from epicardial adipose tissues in the lumen or surrounding tissues. The CT value of calcified components was >130 HU, and that of noncalcified components was <130 HU. According to plaque composition, the coronary lesions of interest were classified as a significantly calcified plaque group , or a nonsignificantly calcified plaque group .Coronary plaques were defined as visible structures >1.0 mm2.3ICA was performed by an experienced interventional cardiologist. A reference guide tube was used for correction, along with an edge detection system for measuring the reference diameter, minimum luminal diameter, length of the lesion, and calculation of the percentage of stenosis. The stenosis was divided into 4 grades as follows: mild stenosis (stenosis percentage <50%), moderate stenosis (50%–69%), severe stenosis (70%–98%), and subtotal-to-total occlusion (99%–100%). Evaluation of thrombolysis in myocardial infarction flow grades during imaging was as follows: Grade 0, no blood perfusion or blood flow at the distal end of the coronary artery; Grade 1, some contrast agent reached the distal end of the coronary stenosis with incomplete filling; Grade 2: the distal end of the coronary artery stenosis could be completely filled, but the image development and elimination of contrast agent were slow; Grade 3: complete and rapid filling and elimination of contrast agent at the distal end of the coronary artery; similar to normal coronary arteries.2.4In case of significant clinical symptoms or ≥50% stenosis was observed on the ICA results, FFR was measured . Prior to measurement, nitroglycerin (100–200 g) was administered into the coronary artery, and then a pressure monitoring guide wire was delivered to the distal end of the stenosis, followed by intravenous or intracoronary injection of adenosine triphosphate (ATP) (140 g/kg/min) to induce a maximum congestive state. FFR was equal to the ratio of mean distal stenosis pressure to mean aortic root pressure at the maximal coronary hyperemia status. FFR≤0.80 was specified as the diagnostic standard for functional stenosis.2.5CT blood flow dynamics modeling and computational fluid dynamics numerical simulation were carried out by an independent core laboratory  blinded to FFR, coronary CTA, and ICA results. The 3 key steps were as follows: first, based on the DICOM image of conventional coronary CTA at the end-diastolic stage, Mimics 10.1 , Geomagic Studio 2014  and SolidWorks 2014  were used to establish an accurate individualized anatomical model of the coronary artery. Second, based on the principle that resting coronary blood flow is proportional to myocardial oxygen demand, the individual boundary conditions at the entrance and exit of the coronary artery were calculated according to the myocardial mass and mean arterial pressure of individual patients. Third, the fluid dynamics Navier Stokes equation  was used for iterative calculation, extraction, analysis, and visualization. The pressure difference (ΔP) between each point of the coronary artery lesions and the entrance of the coronary artery was calculated. iFRCT was defined as the mean pressure distal to the stenosis during the diastolic wave-free period of rest state (Pdwave-free period of rest state) divided by the mean aortic pressure during the same state (Pawave-free period of rest state) (Equation 1), in which Pdwave-free period of rest state was equal to Pawave-free period of rest stateminus ΔP (Equation 2), and thus the contours of iFRCT were obtained. The whole process of modeling and calculation for each case took about 4 hours. iFR2.6CT, thus comparing the correlation difference of FFR and iFRCT between the significantly and nonsignificantly calcified plaque groups. FFR value was used as the gold standard for diagnosis of functional coronary stenosis. The ROC curve was used to select the most suitable cut-off value of iFRCT for the diagnosis of functional coronary artery stenosis, and to investigate the diagnostic efficiency of iFRCT for functional stenosis. The difference between the diagnostic efficiency of iFRCT in the significantly and nonsignificantly calcified plaque groups was evaluated using Fisher test. Coronary CTA luminal stenosis ≥50% was determined as the critical value for the diagnosis of functional stenosis-causing myocardial ischemia. Logistic regression was used to calculate the predictive probability of CTA combined with iFRCT, and then ROC curve analysis was used to investigate the impact of coronary CTA combined with iFRCT on diagnostic efficiency for functional stenosis. The difference of AUC in the diagnosis of functional stenosis using CTA alone and CTA combined with iFRCT was compared using Delong method. P<0.05 was considered statistically significant.SPSS for Windows version 20.0  and MedCalc for Windows version 12.7.7  were used for statistical analysis. All variable data were presented as mean ± standard deviation. Pearson correlation analysis was used to analyze the correlation between FFR and iFR33.1This study included 33 eligible patients with a total of 47 vascular lesions. The demographic and clinical characteristics of the study population are shown in Table 3.2CT value was higher than the FFR value. However, the iFRCT value of the significantly calcified plaque group was lower than the FFR value  0.72–0.95 vs 0.32–0.90, respectively) , with an optimal iFRCT cut-off value of 0.82 , and negative predictive value (NPV) of 70.59%, 83.33%, 70.59%, and 83.33%, respectively.FFR≤0.80 was determined as the gold standard for the diagnosis of functional stenosis-causing myocardial ischemia. AUC for iFR.82 Fig. . At this3.4CT in this group were 94.44% and 83.33%, 100%, 100%, and 92.31%, respectively. There were 29 vascular lesions in the nonsignificantly calcified plaque group, of which lesions with FFR ≤0.80 accounted for 37.93% (n = 11). The diagnostic accuracy, sensitivity, specificity, PPV, and NPV of iFRCT in this group were 68.97%, 63.64%, 72.22%, 58.33%, and 76.47%, respectively. However, there was no significant difference in the diagnostic accuracy of iFRCT between these 2 groups detected by Fisher exact test (P = 0.07).There were 18 vascular lesions in the significantly calcified plaque group, of which lesions with FFR ≤0.80 accounted for 33.33% (n = 6). The diagnostic accuracy, sensitivity, specificity, PPV, and NPV of iFR3.5CT alone, and their combination in the diagnosis of ischemic stenosis is shown in Fig. CT, with its specificity increased to 90% and PPV to 80%. Compared with the AUC when using coronary CTA alone for diagnosis, the diagnostic efficacy of CTA (50%) was significantly higher after introduction of iFRCT , with the AUC value of the ROC curve of 0.90, demonstrating that diagnosis of coronary ischemic stenosis was significantly improved by combining CTA with iFRCT (P = 0.04).FFR≤0.80 was determined as the gold standard for the diagnosis of ischemic stenosis. The efficacy of coronary CTA and iFR4CT and FFR. The application of iFRCT showed high efficiency for the diagnosis of coronary stenosis, and it also had good diagnostic accuracy for functional stenosis caused by significantly calcified plaques. More importantly, using FFR≤0.80 as the gold standard for control, the diagnostic ability of iFRCT combined with coronary CTA for functional stenosis was significantly improved, with higher specificity and PPV compared with using CTA alone.The current study demonstrated a good correlation between iFR found a transient wave-free interval during cardiac diastole (from 25% of the process of diastole to 5 ms before its ending), during which time the measured cardiac microcirculation resistance was naturally constant and minor. This is similar to the average microcirculation resistance of the whole cardiac cycle during maximum cardiac expansion induced by adenosine. iFR is in theory similar to FFR and its result is independent of heart rate, blood pressure, and even blood pressure change caused by ectopic arrhythmia or respiration. The ADVISE study showed that iFR had good reproducibility  and was highly correlated with FFR (r = 0.90). However, the VERIFY study drew different conclusions, pointing out that iFR was influenced by vasodilators and correlated weakly with FFR. In addition, the RESOLVE study and Johnson et al found hyperemic indicators provided diagnostic performance superior to that of resting iFR.,26 Although resistance was reduced further by administration of adenosine, the CLARIFFY study demonstrated that differences in magnitude of microvascular resistance did not influence diagnostic categorization, and iFR, iFRa (adenosine administration), and FFR had equally good diagnostic agreement with HSR. Meanwhile, Petraco et al proved that hyperemic FFR flow was similar to baseline iFR flow in functionally significant lesions . Furthermore, iFR showed a stronger correlation with underlying CFVR  and also agreed more closely with CFVR in stenosis classification . Therefore, iFR can be used as an adenosine-free alternative for FFR with a good diagnostic performance. When 0.83 is used as the threshold for iFR, its diagnostic sensitivity is 91%, with specificity of 85%, PPV of 91%, and NPV of 85%. The ADVISE registry study demonstrated that, based on the inherent variability of FFR, the consistency in critical lesion classification using iFR≤0.89 and FFR≤0.80 as the threshold was as high as 94%, and 81% of the critical lesions with inconsistent classification had associated FFR values located within the FFR “gray zone”(0.75–0.80). Petraco et al used iFR < 0.86 as the threshold (PPV 92%) for percutaneous coronary intervention (PCI), iFR>0.93 as the threshold (NPV 91%) for delay of PCI, and only performed adenosine detection of FFR in patients with iFR0.86–0.93. Such an iFR–FFR hybrid method can reduce the use of adenosine in patients by 57%, and achieve a classification consistency of 95% with FFR. The results of the current study show that iFRCT is significant correlated with the invasively measured FFR, just as in previous studies,,27,28,30 which provides a new evidence supporting the use of iFR as an evaluation index for functional stenosis.To the best of our knowledge, this study is the first blood flow dynamics simulation and calculation of resting state index iFR based on conventional coronary CTA images. Sen et alCT for the diagnosis of coronary artery stenosis. The diagnostic accuracy, sensitivity, specificity, PPV, and NPV were 78.72%, 70.59%, 83.33%, 70.59%, and 83.33%, respectively. Such results are mostly equivalent to the diagnostic efficacy of FFRCT reported in previous multicenter studies. Previous prospective studies, DISCOVER-FLOW, DeFACTO, and NXT have demonstrated diagnostic accuracy, sensitivity, specificity, PPV, and NPV of FFRCT of 73% to 81%, 86% to 90%, 54% to 82%, 65% to 74%, and 84% to 93%, respectively. Our study confirmed the feasibility of using a computational fluid dynamics model for noninvasive detection of the physiological and pathological changes in the coronary artery, thus providing a new noninvasive modeling index. Meanwhile, without the step of simulating maximal blood filling, our new method is closer to the true physiological state during CTA scanning.Our study demonstrated the high efficiency of using iFRCT could increase diagnostic specificity fourfold, PPV by 74.38%, and accuracy by 68.17%. The significant increase in specificity will avoid unnecessary invasive examinations and reduce medical costs. Compared with the diagnostic efficacy of iFRCT alone, combining iFRCT with CTA slightly enhances the diagnostic specificity, PPV, and diagnostic accuracy. Although no significant difference was found between the diagnostic efficacy of iFRCT alone and iFRCT combined with CTA, 2 cases classified as false positive by iFRCT were reclassified as true negative using combined diagnosis. This improved the diagnostic performance of coronary CTA for functional ischemia, showing the potential of coronary CTA combined with iFRCT as the gatekeeper for ICA and revascularization therapy.We have demonstrated that, compared with using a stenosis diameter≥50% as the diagnostic standard for indicating obstructive coronary artery disease, iFR,34 The current study showed good correlation between iFRCT and FFR  in the significantly calcified plaque group, in which iFRCT demonstrated highly accurate diagnostics and specificity of 94.44% and 100%, respectively. Although our study divided cases into different groups based on the number of calcified plaques in the lesion, the results were similar to previous studies using Agatston score. Miyoshi et al analyzed the data of the Japanese NXT study, and reported that FFRCT still had a high diagnostic efficiency (accuracy of 85% and specificity of 81%) for patients with an Agatston score of 400 to 1000. Norgaard et al analyzed 333 lesions in 214 patients and showed that, compared with CTA, FFRCT had a higher diagnostic efficiency for ischemic stenosis in patients with a high Agatston score (416–3599) , and in vessels with a high Agatston score (121–1703) . There are 2 possible reasons for this. First, the cases with severe coronary artery calcification were reconstructed using sharp convolution function reconstruction algorithm, which improved the spatial resolution and the boundary between the vessel lumen and calcified plaques, allowing easier luminal identification in modeling. Second, in addition to correlation with luminal border, hemodynamic simulation of iFRCT also involved other parameters, such as blood pressure, blood viscosity, myocardial mass, and heart rate. This decreased the influence of inaccurate luminal identification on the results, which is unlike the coronary CTA that solely relies on the luminal outline. Such results suggest that iFRCT may be a good auxiliary method for CT in the evaluation of coronary artery stenosis caused by severe calcification. This avoids excluding patients with severe calcification from the indications for CTA examination, providing a new method by which coronary artery disease can be examined without invasive techniques in a high-risk population with severe calcification.Beam hardening effects, halo artifacts (blooming), and partial volume effects could lead to overestimation by CTA of the degree of coronary stenosis caused by calcified plaques.CT threshold value for the diagnosis of functional stenosis was provided, data from a larger sample are needed to verify the results. This study included only patients with chronic coronary artery disease without a history of coronary artery surgery and acute myocardial infarction; therefore, the applicability of iFRCT in patients with PCI, bypass surgery, and acute coronary syndrome is unknown. In this study, FFR≤0.80 was specified as the diagnostic standard for functional stenosis, as with most of the similar studies in recent years.,22,32,37 But FFR is not only influenced by the severity of luminal stenosis, but also by coronary artery morphology and plaque characteristics.–40 Therefore, the diagnosis efficiency of iFRCT for patients with different plaque characteristics and coronary artery morphology needs further validation. The modeling calculation was complicated and time-consuming. Data processing was carried out by independent laboratories, and the simulation and calculation on each patient took about 4 hours, which was affected by the performance of the computer and modeling software used. By upgrading and optimizing equipment and technology, the calculation time is expected to be significantly reduced.However, the current study has the following limitations: the sample size was relatively small. The proportion of patients with positive FFR was only 36.17%, indicating possible selection bias. Because of the small sample size, our study analyzed diagnosis effectiveness based only on blood vessels rather than patients. Although the iFR5CT is strongly correlated with invasive FFR, and has a higher accuracy, specificity, and PPV than CTA in the diagnosis of functional coronary artery stenosis. Combining with iFRCT can improve the diagnostic accuracy of coronary CTA in the diagnosis of functional stenosis, which also has a good diagnostic ability for coronary stenosis caused by lesions with severe calcification. iFRCT is expected to become a noninvasive, adenosine-independent physiologic index for the diagnosis of ischemia-caused coronary stenosis based on resting state coronary CTA; however, additional studies are needed to determine whether iFRCT has the same diagnostic accuracy as FFRCT for the detection of ischemia-causing coronary stenosis.iFRThe authors thank Dr Quanmin Jing , Dr Benqiang Yang , and Dr Haishan Zhang for their help in collecting patient.


Anopheles mosquito midgut harbors a diverse group of endogenous bacteria that grow extensively after the blood feeding and help in food digestion and nutrition in many ways. Although, the growth of endogenous bacteria is regulated by various factors, however, the robust antibacterial immune reactions are generally suppressed in this body compartment by a heme peroxidase HPX15 crosslinked mucins barrier. This barrier is formed on the luminal side of the midgut and blocks the direct interactions and recognition of bacteria or their elicitors by the immune reactive midgut epithelium. We hypothesized that in the absence of HPX15, an increased load of exogenous bacteria will enormously induce the mosquito midgut immunity and this situation in turn, can easily regulate mosquito-pathogen interactions. In this study, we found that the blood feeding induced AsHPX15 gene in Anopheles stephensi midgut and promoted the growth of endogenous as well as exogenous fed bacteria. In addition, the mosquito midgut also efficiently regulated the number of these bacteria through the induction of classical Toll and Imd immune pathways. In case of AsHPX15 silenced midguts, the growth of midgut bacteria was largely reduced through the induction of nitric oxide synthase (NOS) gene, a downstream effector molecule of the JAK/STAT pathway. Interestingly, no significant induction of the classical immune pathways was observed in these midguts. Importantly, the NOS is a well known negative regulator of Plasmodium development, thus, we proposed that the induction of diverged immune pathways in the absence of HPX15 mediated midgut barrier might be one of the strategies to manipulate the vectorial capacity of Anopheles mosquito. Plasmodium and numerous vector control measures are worth mentioning here. However, the worldwide reporting of drug resistance in Plasmodium is also the major challenge in this field  have been identified in anophelines midgut that inhibit Plasmodium falciparum or P. vivax sporogonic development. Mosquitoes treatment with antibiotics reduced the number of midgut bacteria that, in turn, reversed their effect on Plasmodium development  induced against bacteria might kill the parasite  and 12 h light:dark cycles as described before (d before . Larvae Escherichia coli (MTCC 40) and Micrococcus luteus (MTCC 106) bacteria were obtained from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India and used throughout the study. Bacteria were grown separately in LB media following standard protocols (A600 = 0.5) was mixed and centrifuged at 2300 × g for 5 min. The cell pellet was washed twice with Ashburner’s PBS  and finally suspended in 3 ml fresh mouse blood . Overnight starved adult A. stephensi females were allowed to feed on mouse blood supplemented with or without bacterial suspension through an artificial membrane feeder as before (Wild type rotocols . The gros before . After dTable 1) was analyzed through qPCR using Sybr Green Master Mix (Biorad Cat no. 170-8882) and normalized against the constitutively expressed S7 ribosomal protein gene as before  and following manufacturer’s instructions. First-strand cDNA was synthesized using Quantitech reverse transcription kit . The expression of various genes  or AsHPX15 (428bp) was cloned in TOPO 2.1 vector and PCR amplified using M13F, 5′GTAAAACG ACGGCCAGT3′ and T7-M13R, 5′CTCGAGTAATACGAC TCACTATAGGGCAGGAAACAGCTATGAC3′ primers with the following conditions; 94°C for 5 min, 40 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and final extension was carried at 72°C for 10 min. Amplicons were purified with the QIAquick PCR Purification Kit . PCR-purified amplicons tailed with T7 promoter sequences were used to synthesize dsRNA with the MEGAscript kit (Ambion) following the manufacturer’s instructions. For dsRNA mediated interference (RNAi), 1–2 days old female mosquitoes were injected with 69 nl of 3 μg/μl dsLacZ (controls) or dsAsHPX15 (silenced) RNA into their thoraxes using a nanojector . Five days after the injection, mosquitoes were allowed to feed on mouse blood supplemented with or without 10t-test using GraphPad Prism 5.0 software  and Gram- (E. coli) bacteria and the growth kinetics of midgut bacteria was analyzed. Results presented in Figure 1 revealed that the relative levels of 16S rRNA for endogenous bacteria increased 25-fold after 3 h of blood feeding when compared to the sugar fed midguts (p = 0.010). Afterward, the relative levels of endogenous bacteria were 1900, 133, and 270 times after 6, 12, and 18 h of post blood feeding, respectively, against the sugar fed controls . This showed significant reduction in the levels of endogenous bacteria at 12 h (p = 0.0008) and 18 h (p = 0.0011) when compared to the 6 h blood fed midguts. Furthermore, the endogenous bacteria levels increased 7000 times in 24 h blood fed midguts in comparison to the sugar fed midguts . These results are in agreement with previous findings where an increase in endogenous bacteria was observed after 6 h of blood feeding and that further followed the similar growth pattern  bacteria. The majority of these bacteria are Gram-negative and their growth is induced after blood feeding . In para pattern .Figure 1). Interestingly, the relative 16S rRNA levels were indifferent at 6 h (p = 0.0645) and 12 h (p = 0.1696), respectively, when compared to 3 h post bacteria fed midguts. However, the expression levels of 16S rRNA gene at 18 h were reduced fivefold (p = 0.0041) against 3 h post fed midguts . Thus, these altered levels of 16S rRNA at different time points indicated that the growth of bacteria is regulated rhythmically, most probably by the midgut immunity.Parallelly, in case of exogenous bacteria fed midguts, the relative levels of 16S rRNA were 150,000-fold at 3 h in comparison to the sugar fed midguts  to the mosquitoes. The comparative levels of 16S rRNA revealed that the exogenous bacteria fed midguts have 28- and 15-fold higher bacteria load at 6 and 24 h, respectively, when compared to the just blood fed midguts . The reduction in the folds ratio at 24 h seems to be due to the decreased levels of 16S rRNA in exogenous bacteria fed midguts and increased levels of endogenous bacteria in the corresponding blood fed controls. This might indicate that the midgut environment is particularly favorable for the growth of endogenous bacteria in comparison to the exogenous fed microbes.It is noteworthy to mention that in our study, we fed a large amount of bacteria . The induction of AsHPX15 gene in 6 h post bacteria supplemented blood fed midguts was similar to the blood fed controls (p = 0.6485). However, its expression levels were reduced drastically at 12 and 18 h in bacteria fed midguts against blood fed controls (p = 0.0018 for 12 h and p = 0.0038 for 18 h). Interestingly, the relative AsHPX15 mRNA levels were higher in 3 and 24 h post bacteria fed midguts against the blood fed controls. Thus, the heavy load of exogenous bacteria in the midgut had no effect on the early phase of AsHPX15 gene expression . This may be simply due to the regulation of this gene by ecdysone hormone, which is induced in blood fed mosquitoes as reported before . Thus, we hypothesized that some of the immune genes might be controlling the overgrowth of bacteria in these midguts. To test this concept, we analyzed the expression of a heme peroxidase HPX8, which has been reported to be an antibacterial gene in A. gambiae , however, at other time points, HPX8 mRNA levels in the test and controls were indifferent. This induction pattern of HPX8 gene  corroborated with the bacterial 16S rRNA levels in these midguts . These findings indicated that HPX8 might be one of the candidate genes that participate in the midgut antibacterial immunity.Our results revealed that the numbers of exogenously fed bacteria not only increase in the mosquito midgut, it is also regulated at different time points ( gambiae . ResultsTable 1). Our analyses revealed that mRNA levels of GNBP were induced ∼17-fold after 3 h of blood feeding when compared to the sugar fed midguts and that remained unaffected by the presence of bacteria in the blood . However, at 6 and 12 h post bacteria feeding, the relative mRNA levels of GNBP were six and two folds higher, respectively, against the normal blood fed controls . Furthermore, the relative mRNA levels of GNBP were seven folds higher in 24 h post bacteria fed midguts in comparison to the blood fed controls . These findings indicated that GNBP induction represents two episodes, an early event around 6 h and a late phase expression around 24 h post bacteria feeding. Thus, to further explore the immune regulatory mechanism that balances the mosquito midgut immunity against exogenous bacteria, these two time points were further analyzed in detail.In insects, Gram-negative binding protein (GNBP) acts like the pattern recognition receptor (PRR) and triggers classical innate immunity through Toll or Imd pathway against Gram-positive or Gram-negative bacteria, respectively . In anopFigure 3, p = 0.036). However, the induction of this gene was indifferent at 24 h in these midguts (p = 0.563). In addition, we also analyzed the expression of gambicin in these samples. Gambicin is an effector antimicrobial peptide, which is regulated by both Toll and Imd pathways . Although, gambicin expression is further induced in 24 h post bacteria fed midguts, however, these levels were similar to the blood fed controls (p = 0.294). Because gambicin is effective against both Gram+ and Gram- bacteria, therefore, its expression at early and late time points corroborate with the increased number of midgut bacteria . In conclusion, the induction of classical Toll and Imd immune pathways regulate the growth of bacteria in blood fed midguts.We studied the induction of Toll and Imd pathways in the above-mentioned samples those were collected at early 6 h and late 24 h post feeding. The gene expression analyses revealed that the Toll mRNA levels were ∼three folds higher in the 6 h post bacteria fed midguts in comparison to the blood fed controls . Collectively, these results suggested that NOS is not induced in the midgut against exogenous fed bacteria. Thus, we believed that the STAT pathway does not participate in A. stephensi midgut antibacterial immunity and confirms the previous findings as reported in other mosquitoes . These results revealed that the induction of SOCS gene in bacteria fed midguts might be responsible for inhibiting the activation of STAT pathway and NOS expression in a way similar to other systems  gene, which is a downstream feedback repressor of the STAT pathway . Interes systems .M. luteus and E. coli). The midguts were collected at early 6 h and late 24 h post feeding to analyze the expression profiling of the immune genes as mentioned above. Our analysis of AsHPX15 mRNA levels in controls and silenced midguts revealed that this gene was silenced in the range of 80–90% . The relative 16S rRNA levels in controls and silenced midguts were similar at 6 h post feeding (p = 0.053). However, at 24 h post feeding there was 63% reduction in the 16S rRNA levels in the silenced midguts against the unsilenced controls . Thus, AsHPX15 silencing reduces the overall growth of bacteria in the midgut bolus that might include the endogenous as well as exogenous bacteria. These findings are in partial agreement with previous reports where silencing of A. stephensi AsHPX15 ortholog drastically reduced the growth of endogenous bacteria in A. gambiae midguts  and 24 h (p = 0.0111) post bacteria feeding, when compared to their respective unsilenced controls. Thus, the reduced levels of GNBP expression in silenced midguts might reflect the compromised situation for the recognition and induction of classical immune pathways. This fact was further confirmed after analyzing the gene expression of Toll and Imd pathways in these samples. We found that the levels of Toll mRNA were indifferent in silenced and control midguts after 6 h of bacteria feeding . However, Toll mRNA levels were suppressed seven folds in the silenced midguts than controls at 24 h post bacteria feeding (p = 0.0209). These results revealed that Toll pathway is not induced in AsHPX15 silenced midguts neither at early nor at late stage of post bacteria feeding. Furthermore, we analyzed the expression of gambicin in these AsHPX15 silenced samples. We observed that the expression of gambicin gene was significantly downregulated in the silenced midguts after 6 h post feeding against controls . Interestingly, at 24 h post feeding the expression of gambicin gene was induced ∼three folds in silenced midguts in comparison to the controls (p = 0.004). These findings suggested that the induction of gambicin in 24 h silenced midguts might be regulated by some other mechanism(s) that is different from classical Toll and Imd pathways.To understand the immune regulation of bacteria in the AsHPX15 silenced midguts, we analyzed the expression profile of important classical immune pathways. Results presented in Figure 7 revealed that NOS and SOCS genes were induced 15- and 3-folds, respectively, in the silenced midguts after 6 h of bacteria feeding when compared to the controls (p = 0.0002 for NOS and p < 0.0001 for SOCS expression). However, the expressions of both these genes in the silenced midguts were reduced 4- and 1.3-folds, respectively, after 24 h post feeding against unsilenced controls (p < 0.0001 for NOS and p = 0.068 for SOCS expression). This pattern of NOS and SOCS expression indicated the activation of STAT pathway in silenced midguts. Interestingly, the profile of NOS expression also corroborated with the levels of bacteria in the silenced midguts . We believed that the induced NOS during the early hours  might be responsible for reducing the levels of bacteria at later time points  as this gene is reported to play an antibacterial role in mosquitoes as well as other insects . We analyzed the expression of this gene to understand its role in regulating the bacterial load when AsHPX15 mediated physical barrier formation is interrupted. Results presented in Figure 8 revealed that in dsLacZ injected control midguts, HPX8 gene was induced 650-fold after 24 h of exogenous bacteria feeding against sugar fed midguts. However, its expression was reduced ∼40 times in AsHPX15 silenced midguts when compared to the unsilenced controls . Moreover, the expression levels of HPX8 gene in controls and silenced midguts at 6 h post feeding were indifferent (p = 0.078).As, we found before that HPX8, an antibacterial peroxidase, is induced after the exogenous bacteria feeding  and gambicin seem to be the major determinants of the antibacterial immunity in these midguts.Figures 1 and 2). Interestingly, we observed the reduction of AsHPX15 gene expression in 12 h post bacteria fed midguts . This effect might be due to the SOCS mediated suppression of STAT pathway . The presence of STAT binding site in the regulatory region of AsHPX15 gene supported this assumption . It may be due to the selection and growth of specific types of midgut bacteria at certain time points or all types of bacteria have variable growth patterns at different time of post blood feeding. Moreover, these assumptions warrant further investigations. On the other hand, the midgut immunity might also play an important role in regulating the growth of bacteria at certain moments therefore, some of the classical immune genes such as, HPX8, GNBP, Toll and gambicin are induced at 6 h after bacteria supplemented blood feeding . However, in case of bacteria-free blood fed control midguts, the induction of these genes is comparatively minimal to facilitate the growth of endogenous bacteria. Thus, the mosquito midgut system might determine the degree and strength of immune activation on the basis of bacterial or antigenic load in blood bolus. These findings may also explain the previous observations by other researchers where feeding the exogenous bacteria with Plasmodium infected blood reduced the parasite development . These findings are in agreement with the previous study where silencing of AsHPX15 ortholog in A. gambiae suppressed the growth of endogenous bacteria through the induction of bacteria-specific classical immunity . The silencing of AsHPX15 gene and thus, reducing the formation of mucin barrier, might alter the number of bacteria in the midgut. In AsHPX15 silenced midguts the classical immune pathways are mostly ineffective  and the bacteria levels are regulated by other stronger immune mechanisms. Thus, NOS is activated at early hours in AsHPX15 silenced midguts to control the heavy load of bacteria (that includes exogenous as well as endogenous bacteria) . In addition, the induction of SOCS gene in parallel to the NOS might again indicate the activation as well as balanced regulation of JAK/STAT pathway in AsHPX15 silenced midguts . Our findings also revealed that the levels of endogenous bacteria are very less in comparison to the exogenous fed bacteria  thus, NOS is induced in AsHPX15 silenced midguts to control the outsized load of these bacteria . In addition, the induction of gambicin, a classical immune pathway gene, in AsHPX15 silenced midguts might indicate its regulation through JAK/STAT pathway as suggested by other researchers , however, STAT pathway predominates as antibacterial in the absence of AsHPX15 gene expression . This might indicate that the bacterial load and its interaction with the midgut epithelium boosts more effective immune molecule to manage the microbial homeostasis. Thus, we proposed a model as shown in Figure 9 that AsHPX15 helps in the formation of mucin barrier in A. stephensi midgut. This mucin barrier blocks the direct interactions of bolus bacteria with the immune reactive gut epithelial cells. However, the increased growth of bacteria or their elicitors activate the classical Toll and Imd pathways that maintain gut bacterial homeostasis. On the other hand, in AsHPX15 silenced midguts the formation of mucin barrier is compromised and bacterial elicitors activate the midgut epithelial immunity. As a result, midgut epithelial cells mount a strong immune response through the activation of STAT pathway to regulate the growth of midgut bacteria and protecting the mosquito against its deleterious effects .Immune regulation of a huge exogenous bacterial load in the AsHPX15 gene silenced midguts revealed that there is a circumstantial activation of antibacterial immunity. In other words, classical immune pathways are most active against increasing load of bacteria when AsHPX15 gene is expressed (A. stephensi modulates the immune system and co-infection of Plasmodium with exogenous bacteria will result in the negative regulation of the parasite development through induction of specific immune pathways. Because HPX15 is highly conserved gene exclusively among anophelines (Based on our findings, we emphasized that manipulation of AsHPX15 gene in phelines , 2016a, MK, LG, and SK designed experiments for this work. MK, TC, PK, KG, and RD collected samples to perform experiments. MK, TC, PK, KG, RD, LG, and SK performed AsHPX15 and bacterial kinetics experiments and analyzed the induction of different immune genes. MK, TC, PK, LG, and SK performed AsHPX15 silencing experiments. MK, LG, and SK analyzed the data and wrote the manuscript with input from all authors. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saccharum spp.) is one of the most important economic crops because of its high sugar production and biofuel potential. Due to the high polyploid level and complex genome of sugarcane, it has been a huge challenge to investigate genomic sequence variations, which are critical for identifying alleles contributing to important agronomic traits. In order to mine the genetic variations in sugarcane, genotyping by sequencing (GBS), was used to genotype 14 representative Saccharum complex accessions. GBS is a method to generate a large number of markers, enabled by next generation sequencing (NGS) and the genome complexity reduction using restriction enzymes.Sugarcane  filtering. By using the established pipeline, a total of 76,251 non-redundant SNPs, 5642 InDels, 6380 presence/absence variants (PAVs), and 826 copy number variations (CNVs) were detected among the 14 accessions. In addition, non-reference based universal network enabled analysis kit and Stacks de novo called 34,353 and 109,043 SNPs, respectively. In the 14 accessions, the percentages of single dose SNPs ranged from 38.3% to 62.3% with an average of 49.6%, much more than the portions of multiple dosage SNPs. Concordantly called SNPs were used to evaluate the phylogenetic relationship among the 14 accessions. The results showed that the divergence time between the Erianthus genus and the Saccharum genus was more than 10 million years ago (MYA). The Saccharum species separated from their common ancestors ranging from 0.19 to 1.65 MYA.To use GBS for high throughput genotyping highly polyploid sugarcane, the GBS analysis pipelines in 14 Saccharum complex and other related species. A large number of sequence variations were discovered in the Saccharum complex, including SNPs, InDels, PAVs, and CNVs. Genome-wide SNPs were further used to illustrate sequence features of polyploid species and demonstrated the divergence of different species in the Saccharum complex. The results of this study showed that GBS was an effective NGS-based method to discover genomic sequence variations in highly polyploid and heterozygous species.The GBS pipelines including the reference sequences, alignment methods, sequence variant callers, and sequence depth were recommended and discussed for the  Next generation sequencing (NGS) can generate millions of sequencing reads in parallel in a single run, greatly changing the landscape of genetics by determining the genotypes of interest at an unimaginable speed . The effTo capture genome-wide sequence variations and developing genetic markers for species with complicated genomes, it would be feasible to sequence a subset of the genome after reduction of genome complexity. Several methods that utilize NGS to detect sequence variations by sequencing a subset of genomes have previously been discussed . Among tTarget enrichment methods include polymerase chain reaction (PCR)-based target amplification, capture-by-circularization, and hybrid capture methods , 9. In tGBS is a technology enabled by NGS and could reduce genome complexity using restriction enzymes. Compared to the target capture methods, GBS is easy, quick, highly reproducible, and is able to detect genome-wide sequence variations without depending on prior sequence information . GBS hasAlthough these NGS-based methods are popular and powerful, it is daunting to perform them for genetic variation detection in sugarcane due to its large genome size and high heterozygosity, which requires high sequence coverage to resolving sequence variations with different dosages. With continuing efforts from the sugarcane research community, several projects were reported by exploiting the advantages of NGS after reducing genome complexity of sugarcane. For example, in 2013, Bundock et al. had used probe hybridization capture sequencing (HCS) to enrich sugarcane target regions in comparison with the whole-genome shortgun sequencing approach . The resSaccharum complex is a set of interbreeding species including sugarcane’s close relatives (Mukherjee 1957), considered as the primary gene pool leading to modern sugarcane cultivar development. It’s critically important to mine sequence variations, assesse sequence divergence, and identify the sequence variants controlling critical agronomic traits in Saccharum complex, which will help research community better utilize the genetic resources. Moreover, with the development of NGS, it is essential to explore and evaluate different high throughput genotyping methods at this NGS enabled genomics era for future choosing suitable method specifically for a large scale application. The objectives of this research were 1) to establish the pipelines for GBS data analysis in representative and diverse Saccharum complex accessions 2) to call the sequencing variations in the Saccharum complex; and 3) to investigate the sequence features and genetic relationships of the 14 accessions by using genome-wide SNPs. Our results demonstrated that GBS is an effective way to discover genomic sequence variations in highly polyploid and heterozygous species, which will facilitate further genetic and genomic studies for polyploid sugarcane improvement.PstI cutting site. To evaluate the efficiency of two widely used aligners, Bowtie 2 and BWA-backtrack, we aligned 13 million clean reads from Saccharum hybrid CP88–1762 to sorghum genome 3.0 by using Bowtie 2 and BWA-backtrack, respectively. The percentages of uniquely mapped reads were 33.3% for Bowtie 2 and 13.8% for BWA-backtrack. Bowtie 2 generated much more uniquely mapped reads and was more suitable as the aligner for downstream analyses for sugarcane  remained as high quality clean reads with one of the barcodes and the cane see . UniquelPstI digestion of the sorghum genome 3.0  / [ * (length of the region)]. Then log2 ratio of normalized reads of each two accessions were calculated and used to assess the CNVs. The targeting regions were considered as CNVs with log2 ratios falling outside of the 99% range (mean ± 3sd) of log2 ratio.Only sequence regions with The number of reads for alleles at a SNP locus follows a binomial distribution . Thus, fP = the probability to obtain k reads for allele ‘A’. Based on this formula, the sequence depth was estimated in order to obtain the expected number of reads for minor alleles of SD SNPs with a probability higher than 95% at different ploidy levels. For simplicity of the estimation, we assumed no sequencing error in the clean reads.Where n = the total of sequencing reads, x = the frequency of allele ‘A’, k = the number of reads for allele (A), and Uniquely mapped reads of accession, CP88–1762, were used to determine the relationship of sequence depth and percentage of SNPs called. The data set with the uniquely mapped reads was considered as a whole sequence set that could call all potential SNPs. Eight sub-data sets with 20, 30, 40, 50, 60, 70, 80, and 90% of the whole sequence set were randomly sub-sampled for 10 times, separately. Then SNPs were called in these 10 times of eight sub-sampled data sets. Compared with the whole sequence set, the percentages of SNPs called were calculated for each of the eight sub-sampled data sets.https://probes.pw.usda.gov/batchprimer/) from sorghum genome harboring the SNPs. Only the primers with one alignment to the sorghum genome 3.0 [A total of 16 SNPs called with a minimum sequence depth of 56 per locus per sample Table  were rannome 3.0  and alignome 3.0  with lesConcordantly called SNPs by using GATK and Freebayes with a minimum depth of 56 were used to calculate the percentages of heterozygosity, SD SNPs, multiple allelic SNPs, and transition/transversion (Ts/Tv) ratios for each of the 14 accessions respectively. The Ts/Tv ratio was determined by SnpEff (v4.1) . The perConcordantly called SNPs by five reference-based callers with a minimum depth of 56 were used for phylogenetic analysis of the 14 accessions. A divergence time tree for the 14 accessions and sorghum was constructed using the molecular evolutionary genetic analysis software version 6.0 (MEGA 6.0) , 63. Fir


By contrast, for the sulcal depth, the average number of WMNs per 0.21 mm2 was 41 for FR, 31 for cingulate sulcus (underlying the SFG), 54 for Par and 63 for TE cortical regions. Since at least some WMNs participate in cortical circuitry, these results raise the possibility of their differential influence on cortical circuitry in the overlying gyral and sulcal locations. The results also point to a possible role of WMNs in the differential vulnerability of gyral vs. sulcal regions in disease processes, and reinforce the increasing awareness of the WMNs as part of a complex, heterogeneous and structured microenvironment.Gyrencephalic brains exhibit deformations of the six neocortical laminae at gyral crowns and sulcal depths, where the deeper layers are, respectively, expanded and compressed. The present study addresses: (1) the degree to which the underlying white matter neurons (WMNs) observe the same changes at gyral crowns and sulcal depths; and (2) whether these changes are consistent or variable across different cortical regions. WMNs were visualized by immunohistochemistry using the pan-neuronal label NeuN, and their density was quantified in eight rhesus monkey brains for four regions; namely, frontal (FR), superior frontal gyrus (SFG), parietal (Par) and temporal (TE). In all four regions, there were about 50% fewer WMNs in the sulcal depth, but there was also distinct variability from region to region. For the gyral crown, we observed an average density per 0.21 mm The structural and connectional heterogeneity of cortical sulcal and gyral regions is well known in the context of laminar deformation Welker,  and, morFrom qualitative observations alone, it is obvious that WMNs are more numerous near the gyral crowns than in the sulcal depth. In the present study we addressed the degree to which the population of underlying WMNs observes the same deformations, and whether there are region-specific differences in WMN density in the two locations. Such differences in WMN density could functionally impact circuitry in the overlying cortical gray matter and also impact WM tractography , temporal (TE) and parietal (Par) cortical regions. Average WMN density was similar for TE and Par regions, but lower in FR. No significant density differences were observed between a group of four young adult (6.0–7.8 years old) and four older monkeys (20.5–28.7 years old) suggesting that WMNs, like those in the cortical gray matter, are stable across age in the adult , as better conforming to sulcal and gyral topography. In order to achieve wider representation of association areas, we added a comparison between the cingulate sulcus and the overlying superior frontal gyrus (SFG). In brief, we found that for all four cortical regions, the sulcal density of WMNs was about 50% that at the gyral crown; but in both gyral and sulcal locations, WMN density was least for the SFG region and greatest for the TE.The present investigation compares WMN density specifically in the gyral crowns and sulcal depths of the same eight monkeys Figure . Here, wMacaca mulatta) that were the basis for our previous study . All procedures were approved by the BUMC Institutional Animal Care and Use Committee.The present investigation utilized tissue from the eight male rhesus monkeys , deeply anesthetized with sodium pentobarbital , and euthanized by exsanguination during transcardial perfusion with 4% Krebs buffer at 4°C, followed by 4% paraformaldehyde in phosphate buffer  at 37°C. Brains were blocked 2O2. Sections were washed, mounted onto gelatin-coated slides, air dried and cover-slipped with Permount mounting medium .As described more fully in Mortazavi et al. , sectionAs an extension of Mortazavi et al. , this stThe nomenclature for gross anatomical, macro WM subdivisions is much less well established than that for the overlying gray matter or for defined axon tracts within the WM. For gyral WM regions, in particular, nomenclature sometimes adheres to gross anatomical distinctions  but other times follows gray matter architectonic subdivisions (“area TEd”). In this study, we used “frontal region” to denote the territory from the anterior tip of the principal sulcus to the arcuate spur; the “parietal region”, the zone from the anterior tip of the intraparietal sulcus (IPS) to the posterior part of the superior temporal sulcus (STS); and the “temporal region”, from the mid-amygdala to the anterior IPS. For the temporal region, counts were subdivided so as to correspond to the STG and the gyrus ventral to the STS . Polygons approximated a semi-circle or curved cone for gyral crowns, and crescent for sulcal depths . One edge of this rectangular counting frame was placed at the gyral crown, and four underlying zones (each 1.25 mm) were demarcated medially toward the sulcal depth. The rectangle was placed approximately in the middle of the gyral core, so as to avoid possible overlap with domains associated with gyral walls.In our previous investigation, we found no change in WMN numbers between young adult and aged monkeys . To determine if there was a statistically significant change in the WMN density in moving from the gyral crown towards deep WM, a repeated-measures ANOVA was used. All data are reported as mean ± SEM, and alpha was set at 0.05.2) than in sulcal depths  = 58.41, p < 0.05).Qualitative observations suggest that WMNs are distinctly more numerous in the gyral crowns than sulcal depths Figure . To confF = 24.93, p < 0.05). For gyral locations, WMN density ranged from a low of 57 ± 4 neurons per counting frame in the SFG region to 93 ± 6 WMNs per counting frame in TE . For FR and Par, we found 81 ± 5 and 80 ± 6 WMNs per counting frame, respectively. For sulcal locations, WMN density showed the same trend; that is, least for the SFG/cingulate region (31 ± 4 WMNs per counting frame), intermediate for FR (41 ± 3 WMNs) and Par (54 ± 2 WMNs), and highest for TE . Additionally, post hoc analysis showed that there are significantly more WMNs in both TE and Par  than in FR. While the ratio of sulcal/gyral WMNs varied in the different regions (FR (0.59), SFG (0.59), Par (0.73) and TE (0.70)), the differences were not statistically significant . There is a small reduction from zone 2 to 3 but otherwise numbers are stable from zone 3 to 4; namely, for zone 3, 12 ± 1 neurons in TEd and 22 ± 2 in STG; and for zone 4, 10 ± 1 in TEd and average 21 ± 3 in STG. Statistical analysis confirmed that the drop in WMN density was significant in both STG and TEd, proceeding from the gyral crown medially, towards deep WM  = 98.464, p < 0.05). Further, post hoc analysis showed that there are significantly more WMNs in zones 3 and 4 of STG as compared to TEd . For these two temporal subregions, zone 1 (1.125 mm subjacent to the crown) contained the most WMNs, and zone 2 (the next 1.125 mm) consistently had fewer neurons. While there was overall a progressive fall-off in density across the four zones (of about 50% from zone 1 to zone 4), there were also individual sections where zones 3 or 4 showed a localized increase, or where zones 2–4 were relatively uniform. In the distal portion of the gyrus (furthest from the pia), the density of gyral WMNs is close to the average density of WMNs in the deep WM , 39–84 neurons or 38–94 neurons, respectively in STG and TEd. Loup et al.  nearest the gyral crown, where WMNs typically exhibit a pattern of quasi-regular neuron-sparse circles, 120–250 μm in diameter. Similar neuron-free microzones have been reported in human cortical tissue  has been actively discussed in the context of cortical development (see Lewitus et al., An increased density of WMNs has consistently been associated with schizophrenia (Eastwood and Harrison, in vivo imaging field, the segmentation of gray and WM, and in particular the accurate determination of gray matter/WM borders can have major consequences; for example, MR studies commonly assess thickness of cortical gray matter and WM in disease and normal development and aging. Currently, there is no one algorithm acknowledged as the most accurate because segmentation in MR is largely limited by partial volume effects (volume averaging of voxels; Yeh and Tseng, Finally, as observed in Reveley et al. , the difThe density of WMNs at gyral crowns is about 50% greater than at sulcal depths, but there is regional variation. This result raises the possibility that WMNs may have a differential influence on the circuitry and disease vulnerability of the overlying cortex at these specialized locations. Our results reinforce the fact that the subcortical WM is not homogeneous, but rather is a complex microenvironment, consisting of differential arrangements of WMNs and, presumably, glia, among the WM bundles.FM and KSR wrote the manuscript, designed and conducted immunohistochemical experiments, analyzed the data and prepared illustrations. SER contributed to cell counts, digitized the images and assisted with data analysis and figure preparation. DLR guided the overall experimental design and participated in manuscript preparation.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.


The orchestration of orienting behaviors requires the interaction of many cortical and subcortical areas, for example the superior colliculus (SC), as well as prefrontal areas responsible for top–down control. Orienting involves different behaviors, such as approach and avoidance. In the rat, these behaviors are at least partially mapped onto different SC subdomains, the lateral (SCl) and medial (SCm), respectively. To delineate the circuitry involved in the two types of orienting behavior in mice, we injected retrograde tracer into the intermediate and deep layers of the SCm and SCl, and thereby determined the main input structures to these subdomains. Overall the SCm receives larger numbers of afferents compared to the SCl. The prefrontal cingulate area (Cg), visual, oculomotor, and auditory areas provide strong input to the SCm, while prefrontal motor area 2 (M2), and somatosensory areas provide strong input to the SCl. The prefrontal areas Cg and M2 in turn connect to different cortical and subcortical areas, as determined by anterograde tract tracing. Even though connectivity pattern often overlap, our labeling approaches identified segregated neural circuits involving SCm, Cg, secondary visual cortices, auditory areas, and the dysgranular retrospenial cortex likely to be involved in avoidance behaviors. Conversely, SCl, M2, somatosensory cortex, and the granular retrospenial cortex comprise a network likely involved in approach/appetitive behaviors. The dorsal surface of the skull was exposed and prepared for a craniotomy. Craniotomies (0.7 mm) in positions overlying injection sites were made using a microbur (0.7 mm) and a microdrill.2.1.1A two‐barreled iontophoresis pipette with a tungsten microelectrode (tip 10–20 microns)  , the pipette was left for 20 min after the injections before removing it to allow for optimum diffusion of tracer into the tissue.After a 3–4 days recovery period, the mice underwent a cardiac perfusion. They were given terminal anesthesia of pentobarbital (0.3 ml 200 mg/ml i.p.). Then they were perfused, with a preliminary injection of 1 ml heparin sulfate  Hayat, , followe2.22.2.1Coronal free floating sections (40 µm) were taken and placed in 4% PBS. This was followed by an initial autofluorescence quenching step  and PBS washes (3 × 10 min). Sections were then mounted onto microscope slides with a propidium iodide (PI) medium  or a DAPI medium .2.2.2Coronal free floating sections (40 µm) were taken and placed in 4% PBS. After an initial autofluorescence quenching step (as for retrograde tracing), sections were incubated for 2 hrs in streptavidin‐Alexa 488  , at an excitation wavelength of 350 nm to illuminate endogenous FG fluorescence. Excitation at 530 nm was utilized to highlight nuclei with the PI staining and co‐locate with the tracer signal. Digital images were acquired using “MicroFire” optics.Sections from the anterograde tracing, which had undergone immunohistochemical amplification were examined under a fluorescence microscope . Projection patterns were visualized with excitation at 500 nm; nuclei counterstains were visualized with either 530 nm excitation (PI) or 350 nm (DAPI). Photo‐merges were taken of stained areas for further qualitative and quantitative analysis using AxioVision software. For illustrative purposes photomicrographs were processed for brightness and contrast and gray‐scaled using Adobe Photoshop CS6.2.4Lthresh) according to the following formula:Lmean corresponds to the mean luminance across the region of interest (ROI), and Lσ2 corresponds to the variance of the luminance across the ROI. The ROI chosen for the luminance thresholding was taken from nonlabeled regions of the photomicrograph. Thresholding produced a binary image, where values of 1 displayed the extent of tracer injection. From these images, a contour outlining the extent of labeling was produced by demarcating the limits of the binary signal. These contours were then imported into a vector graphics program and transposed onto representative brain atlas slides  to remove background luminance and were thresholded. This was achieved through custom scripts which calculate the thresholding value  according to the following formula:Lmean corresponds to the mean luminance across the region of interest (ROI), and Lσ2 corresponds to the variance of the luminance across the ROI. As described previously, ROIs selected for thresholding were placed on areas which had no clearly labeled cells or fibers. ROIs for cell counting and fiber label assessment were defined and demarcated on nuclear counterstained images  using the mouse brain atlas as reference  corresponds to the quantified amount of tracer in a particular region of interest, and Q(ROIb) corresponds to the quantified amount of tracer in a complementary region. A preference in connectivity for ROIa would yield a positive number between 0 and 1, a preference for ROIb would yield a negative number between 0 and 1. The code for all of the analysis is available online . Significant differences between the MIs for the particular injection site were tested by a Mann–Whitney U test, alpha value = 0.05.Preferential connectivity of a particular injection site to different ROIs was determined by calculating the modulation index (MI) of connectivity which was calculated as:3We injected the retrograde tracer FG iontophoretically into the SCm or SCl, and we injected the anterograde tracer BDA into the two main cortical SC input structures which are assumed to be key structures involved in top–down behavioral control, namely the Cg or M2. We found that the intermediate and deep layers of the SCl and SCm showed a segregation with respect to specific cortical and subcortical afferents. Moreover, Cg and M2 showed equally substantial segregation regarding their projection sites. The specificity of these connections supports the hypothesis that the medial SC and the Cg are involved in avoidance (aversive) behavior, while SCl and M2 are involved in approach (appetitive) behavior. We will first describe the results from the experiments where retrograde tracers have been injected into the SC, and then describe the experiments where anterograde tracers have been injected into M2 and Cg, respectively.3.1We performed five medial and four lateral injections for retrograde tracing in the mouse SC. Local spread of tracer in all of these cases was confined to the target sites in the SC, that is, lateral injections did not spread into medial parts and vice versa. The injections also did not spread into neighboring brain areas such as the periaqueductal gray (PAG) or the mesencephalic reticular formation (mRt) Figure a–c. Retr3.2Retrogradely labeled cell populations in the neocortex, after injection into the two different subdivision of the SC, were remarkably segregated. As expected, retrogradely labeled cells in the cortex were confined to layer 5b.The secondary visual cortex  Figure a, the prConversely, the somatosensory areas, specifically S1, the barrel field  Figure a, the flIf we take into account neuronal labeling generalized across the entire experimental cohort there was a separation of labeled RSD cells found after SCm injections and RSG after SCl injection, respectively. However, labeled RSG neurons were nevertheless found in two of the six SCm injection cases.p = .016, Mann–Whitney U test, Figure Retrogradely labeled cells after SCm and SCl injections were found in the M2 , and in the Cg . While these two areas showed retrogradely labeled cells after both, SCl and SCm injections, they did so to different degrees. The SCm injections resulted in higher numbers of labeled cells in the Cg Figure d. Conver3.3Regions with retrogradely labeled cells only after SCm injections included the subthalamic nucleus , the dorsal raphe , the external cortex of the inferior colliculus , the parabigeminal nucleus , and the pontine nucleus .p = .016, Mann–Whitney U test) preference for the ventromedial SNR to show retrogradely labeled cells following SCm injections and for the dorsolateral SNR to show retrogradely labeled cells following SCl injections  showed retrogradely labeled cells exclusively after SCl injections. A number of midbrain regions contained retrogradely labeled neurons after injections of tracer into either subdivision of the SC. These included the lateral lemniscus , the PAG , the mRt , the substantia nigra , and the SC . The ll and the PAG showed similar density of retrogradely labeled cells, regardless of the injection site. The SC, mRt and SNR had differential numbers of retrogradely labeled cells following injection into the two subdivisions of the SC. The contralateral SCl was retrogradely labeled following injections into the SCm and the SCl. The mRt  showed a higher number of retrogradely labeled cells after SCl than SCm injections. The SNR equally showed larger numbers of retrogradely labeled cells following SCl injection when compared to SCm injections. In addition, there was a significant  and the ventromedial hypothalamic nucleus .SCl injections did not result in exclusive retrograde label in the thalamus or hypothalamus. A number of thalamic and hypothalamic regions contained retrogradely labeled neurons after both SCm, and SCl injections. The zona incerta ventral part  and dorsal part  displayed retrograde neuronal labeling after injection into SCm and SCl. The ZIV was more strongly connected to the SC (l and m) than the ZID. Moreover, the neuronal projections from the ZI were spatially segregated, with the population projecting to the SCm being located in the dorsolateral region bordering on the dorsal lateral geniculate nucleus (DLG). The population projecting to the SCl was found in the ventromedial portion of ZI Figures f and 5f.3.5The pretectal area  was retrogradely labeled only after SCm injections.Retrogradely labeled cells were found in the ipsilateral nucleus of the posterior commissure  after both SCm and SCl injections, while the contralateral PCom only sends efferents to the SCl.To provide a general overview of input to the SC from the entire brain, we generated a connectivity diagram of the areas which exhibited retrogradely labeled cells after SCm and SCl injections, respectively, Figure .3.6We performed five M2 and four Cg injections with the anterograde tracer BDA. The tracer in all cases was confined to the target area and did not leak into neighboring brain regions such as the corpus callosum (cc) and the third ventricle Figure a–c. We w3.7The prefrontal cortex, the orbital cortex, lateral  and ventral  showed anterograde label exclusively after M2 injections. Anterograde label following M2 injections was found in virtually all primary somatosensory areas with stronger label in the barrel field  Figure a, than tIn addition the ipsilateral primary motor cortex . V2ML was the only sensory area with exclusive anterograde label after Cg injections , the claustrum , the primary visual cortex , the V2MM , the prelimbic cortex , the medial orbital cortex , RSD . Moreover, M2 injections resulted in anterograde labeling in the upper layers of RSD .Midbrain areas with anterograde label after Cg, but not M2 injections, were the ECIC, , the STh , the interpeduncular nucleus , the paramedian raphe nucleus , the median raphe nucleus , and the Pn .Anterograde label in the midbrain after both M2 and Cg injections, was found in the cerebral peduncle , the SNR , the substantia nigra pars compacta , the dorsolateral and ventrolateral PAG , mRt , the SCl , and SCm .Despite the fact that the above areas showed anterograde label after either injection, some areas showed a spatial preference of anterograde labeling within their subdivisions. The PAG was more strongly labeled in the dorso‐lateral part (DLPAG) after Cg injections, while it was more strongly labeled in the ventro‐lateral part (VLPAG) following M2 injections. The substantia nigra, while receiving input from both areas, did so in a topographically biased manner. The SNR received connections from both the Cg and M2 which terminated onto the ventromedial part of the area. The SNC received sparse connections from the Cg and more abundant connections from M2.p = .016) , the lateral septal nuclei , the diagonal band, vertical limb , and the diagonal band, horizontal limb  showed anterograde label. The HDB connections expressed a bias for ipsilateral over contralateral connectivity.3.10The globus pallidus  was anterogradely labeled only after M2, not after Cg injections. The core of the nucleus accumbens  received low levels of input from Cg, but no input from M2.p = .016, Mann–Whitney U test)  was more strongly labeled after M2 injections. Conversely, the dorsomedial striatum  was more strongly labeled following Cg injections Figures d and 10d3.11Anterograde labeling was observed only after M2 injections in the lateral posterior thalamic nucleus, laterorostral part , the central medial thalamic nuclei , and the lateral habenular nucleus  and ventrolateral , the submedius thalamic nucleus , the reticular nucleus , the zona incerta, dorsal  and ventral  portions, the ventromedial thalamic nucleus , the central lateral nucleus  following Cg injections, but not M2 injections.3.13The anterior pretectal nucleus  showed anterograde label following Cg and M2 injections.4We delineated the main cortical and subcortical inputs to the medial and lateral SC of the mouse, as well as the target areas of two key frontal areas providing strong preferential input to these SC subdivisions.We found limited overlap in the cortical and subcortical afferents to the SCm and SCl. The majority of regions which project to the SCm have visual, extra‐personal (far) space and negative affective state related functionality. The majority of regions which project to the SCl have somato‐motor, peri‐personal (near) space related functionality. Areas which were labeled after injection into either of the two subdivisions of the SC, often showed topographically segregated cell populations with limited spatial overlap.The main prefrontal areas providing segregated inputs to middle and lower layers of the SC, Cg, and M2, equally target functionally segregated networks. Areas which received input solely from the Cg are functionally related to vision, emotional state and avoidance behaviors. Areas which received input solely from M2 are functionally related to somato‐sensation, gustation, and approach behaviors. Areas which received projections from both Cg and M2 often had a tendency to have topographical segregation, suggesting that functional specialization in these areas exists at the level of subpopulations.4.14.1.1Our retrograde tracing data are largely consistent with the existing literature , temporal cortical areas , the amygdala and the hippocampus.Brain regions found to connect only to the SCl in the Oh et al.  paper, b4.2In general, the projections identified from Cg and M2 mouse cortical and subcortical targets are similar to those found previously in the rat  to those secondary visual cortical areas. Due to the increased focus in the literature on functionally defined areas it is important to relate anatomically defined label to these functional terms , rostrolateral area (RL), and posteromedial area (PM) , rostrolateral area (RL), and PM  (Ahmadlou & Heimel, 4.4.2The SCl is retinotopically mapped to the lower visual space, where appetitive stimuli, such as prey or offspring are likely to occur, both of which require approach‐orienting responses, (Ahmadlou & Heimel, Although we have highlighted an existing dichotomy in the separation of approach and avoidance behaviors regarding the location of stimuli in the visual field, it must be noted that this segregation is not complete. Studies have used visually stimuli in the upper visual field which require approach behaviors (Harvey, Collman, Dombeck, & Tank, 4.4.3M2 and Cg innervate different sections of the SC. This suggests that they control separate types of orienting behavior. If so, it should be reflected in their cortical and subcortical efferent projections. We investigated this by anterograde tract tracing, and indeed uncovered a difference in projection patterns.M2 mostly sends efferents to SCl and somatosensory cortical areas. M2 in the mouse may be the homolog to FOF in rats (Erlich et al., The Cg is the major source of prefrontal input into the intermediate and lower layers of the SCm. Behaviorally, it has been implicated in top–down modulation of aversion related behaviors. Lesions of the Cg in rabbits reduces avoidance behaviors in relation to noxious stimuli (Gabriel, Kubota, Sparenborg, Straube, & Vogt, In conclusion, our study has revealed anatomically segregated circuits in the mouse brain that likely orchestrate approach and avoidance behavior, respectively. Avoidance behavior is likely subserved by Cg, secondary visual cortices, auditory areas, and the dysgranular retrospenial cortex in conjunction with SCm. Conversely, approach/appetitive behaviors is likely sub‐served by M2, somatosensory cortex, and the granular retrospenial cortex in conjunction with the SCl.The authors declare they have no competing financial interests.