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To date, the only intervention that has cured HIV infection has been bone marrow transplants from HIV-resistant donors to HIV-infected recipients. This approach has been used to both cure hematological malignancies and HIV infection, but it cannot be widely adopted due to the high risk of mortality associated with cell transplants between individuals. To overcome this limitation, several approaches have been developed to generate HIV resistance using gene therapy in an infected individual's own cells. With the growing arsenal of effective methods to generate HIV-resistant cells, a safe and effective combination gene therapy approach to cure HIV infection is fast approaching. Here, we review several gene therapy-based methods to generate HIV-resistant cells including the expression of antiviral genes, genome editing, and transcriptional gene silencing. Their varied mechanisms, advantages, and disadvantages are discussed, and perspectives are provided for how they may be combined to design an effective gene therapy for HIV.

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The RNA-targeting CRISPR nuclease Cas13 has emerged as a powerful tool for applications ranging from nucleic acid detection to transcriptome engineering and RNA imaging1-6. Cas13 is activated by the hybridization of a CRISPR RNA (crRNA) to a complementary single-stranded RNA (ssRNA) protospacer in a target RNA1,7. Though Cas13 is not activated by double-stranded RNA (dsRNA) in vitro, it paradoxically demonstrates robust RNA targeting in environments where the vast majority of RNAs are highly structured2,8. Understanding Cas13's mechanism of binding and activation will be key to improving its ability to detect and perturb RNA; however, the mechanism by which Cas13 binds structured RNAs remains unknown9. Here, we systematically probe the mechanism of LwaCas13a activation in response to RNA structure perturbations using a massively multiplexed screen. We find that there are two distinct sequence-independent modes by which secondary structure affects Cas13 activity: structure in the protospacer region competes with the crRNA and can be disrupted via a strand-displacement mechanism, while structure in the region 3' to the protospacer has an allosteric inhibitory effect. We leverage the kinetic nature of the strand displacement process to improve Cas13-based RNA detection, enhancing mismatch discrimination by up to 50-fold and enabling sequence-agnostic mutation identification at low (<1%) allele frequencies. Our work sets a new standard for CRISPR-based nucleic acid detection and will enable intelligent and secondary-structure-guided target selection while also expanding the range of RNAs available for targeting with Cas13.

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To date, the only intervention that has cured HIV infection has been bone marrow transplants from HIV-resistant donors to HIV-infected recipients. This approach has been used to both cure hematological malignancies and HIV infection, but it cannot be widely adopted due to the high risk of mortality associated with cell transplants between individuals. To overcome this limitation, several approaches have been developed to generate HIV resistance using gene therapy in an infected individual's own cells. With the growing arsenal of effective methods to generate HIV-resistant cells, a safe and effective combination gene therapy approach to cure HIV infection is fast approaching. Here, we review several gene therapy-based methods to generate HIV-resistant cells including the expression of antiviral genes, genome editing, and transcriptional gene silencing. Their varied mechanisms, advantages, and disadvantages are discussed, and perspectives are provided for how they may be combined to design an effective gene therapy for HIV.

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MR angiography of the inflow vessels has undergone dramatic change in the past few years; the advent of ultra fast gradient echo pulse sequences combined with the gadolinium chelate bolus technique has supplanted earlier methods. However, owing to the highly variable flow velocities and inconsistent rate of vessel opacification encountered in the outflow vessels, two-dimensional time-of-flight MR angiography remains the workhorse of the distal vasculature.

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BACKGROUND: Successful management of patients with peripheral vascular disease requires detailed vascular imaging, usually performed by contrast arteriography. Recently, magnetic resonance angiography (MRA) has been shown to be a noninvasive technique with greater sensitivity than contrast arteriography for detecting distal runoff vessels in patients with peripheral arterial occlusive disease. However, to supplant the need for contrast arteriography and provide a completely noninvasive evaluation of patients with occlusive disease, accurate imaging of the inflow vessels and the runoff vessels is necessary. METHODS: We used both conventional arteriography and MRA in preoperative studies of the aorta, iliac, and femoral vessels of 47 patients. Conventional arteriography and MRA studies were compared for their ability to detect vessel patency and the presence of hemodynamically significant stenoses. Independent interventional plans were developed based on the information provided by each technique. The findings of conventional and MRA studies were verified by intraoperative arteriography or direct operative exploration. RESULTS: Results of the two studies were identical in 41 (87%) of 47 patients or 600 (98%) of 614 segments imaged. MRA accurately detected patent and occluded arterial segments (sensitivity 99.6%, specificity 100%, positive predictive value 100%, negative predictive value 98.6%) and hemodynamically significant stenoses. Therapeutic plans based on either MRA or conventional arteriography were identical for each patient. CONCLUSIONS: MRA provides comparable results to contrast arteriography in the proximal arterial system and superior results for imaging the distal vasculature. This noninvasive technique may replace contrast arteriography in a large number of patients in the future.

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PURPOSE: Contrast venography is the gold standard for diagnosis in deep venous thrombosis (DVT); however, this technique is invasive and requires the use of potentially hazardous contrast agents. Although duplex Doppler ultrasonography is accurate in the evaluation of lower extremity DVT, it is less accurate in the assessment of the pelvic and intraabdominal veins. Magnetic resonance venography (MRV) has recently been developed, and our purpose was to determine whether MRV could accurately demonstrated DVT when compared with duplex scanning and contrast venography. METHODS: Eighty-five patients underwent contrast venography and MRV from the inferior vena cava to the popliteal veins to rule out DVT. Thirty-three of these patients also underwent duplex scanning. Blinded readings of these studies were compared for the presence or absence and extent of venous thrombosis. RESULTS: DVT was documented by contrast venography in 27 (27%) venous systems. Results of MRV and contrast venography were identical in 98 (97%) of 101 venous systems, whereas results of duplex scanning and contrast venography were identical in 40 (98%) of 41 venous systems. All DVTs identified by contrast venography were detected by MRV and duplex scanning. The discrepancies were due to false-positive MRV (3) and duplex scanning (1) results. When compared with contrast venography, MRV had a sensitivity of 100%, specificity of 96%, positive predictive value of 90%, and negative predictive value of 100%. For duplex scanning the sensitivity was 100%, specificity was 96%, positive predictive value was 94%, and negative predictive value was 100%. CONCLUSIONS: It is concluded that MRV is an accurate noninvasive venographic technique for the detection of DVT.

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Reconstructive vascular surgery is the last resort for limb salvage in patients with vascular insufficiency. Planning the sites of arterial bypass grafts depends on an exact knowledge of the angiographic anatomy. Currently, conventional contrast angiography is the primary preoperative study for surgical planning. Recently, magnetic resonance (MR) arteriography has been shown to be an accurate technique for the evaluation of slow arterial flow in the lower extremities. The purpose of this review will be to show how MR arteriography can be used as both an adjunct to and in some cases instead of conventional angiography for the preoperative assessment of lower-extremity vascular reconstruction. The derivation of common artifacts, the many pitfalls of interpretation, and the evaluation of peripheral arterial occlusion and percentage stenosis will be discussed. MR arteriography of the lower extremities is an important advance for the noninvasive evaluation of disease of the peripheral vasculature.

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Magnetic resonance angiography (MRA) has recently been shown to be more sensitive than contrast arteriography in the detection of patent distal runoff vessels. This study compares MRA and contrast arteriography in evaluating the severity of stenotic lesions in peripheral arteries, which has not been previously investigated. Forty-eight arterial stenoses (19 patients) were identified, from the distal aorta through the crural vessels. Contrast arteriograms (anterioposterior projection) and MRA axial images were used to measure stenoses. Interobserver agreement of arteriogram readings was excellent (average weighted k = 0.87). Measurements of degree of stenosis as evaluated by MRA and contrast arteriography were analyzed by linear regression and Spearman rank correlation, which showed a high degree of correlation between the two diagnostic modalities (r = 0.83, p < 0.001; rs = 0.84, p < 0.001). These observations suggest that MRA is accurate in the evaluation of peripheral arterial stenosis when compared with the "gold standard" contrast arteriogram. In addition, MRA cross-sectional images provide information beyond that of conventional arteriography, showing details of plaque eccentricity and vessel wall characteristics. In the future, MRA may supplant diagnostic contrast arteriography for many patients.

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When failure of vein grafts is due to technical error it is usually observed in the early postoperative period. In this review we describe late failure of five bypass grafts as a result of entrapment of the vein graft caused by an improperly placed tunnel from the femoral to the popliteal artery. Vein graft entrapment may either produce no symptoms or eventually lead to limb ischemia. Pulses and pressures that vary with flexion and extension maneuvers should alert the clinician to the possibility of an entrapment syndrome. The characteristic arteriographic findings observed in these patients include an aberrant course of the vein graft outside the anatomic course of the popliteal artery and position-dependent compression of the graft. The ability of magnetic resonance angiography to demonstrate the arteriographic appearance of the graft as well as the precise location of the compression and to identify structures involved in the entrapment, make it a valuable noninvasive tool in the preoperative assessment of these patients. Treatment options include division of the gastrocnemius muscle, division of the vein graft, or replacement of the vein graft. Familiarity with vein graft entrapment should facilitate its recognition. Careful placement of bypass grafts along the anatomic course of the popliteal artery within the popliteal space will prevent this complication.

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In the first phase of this study, seven healthy subjects underwent examination with two-dimensional time-of-flight (TOF) magnetic resonance (MR) angiography to develop a protocol for evaluation of peripheral arterial vasculature from the infrarenal aorta to the foot. In the second phase, 73 patients with symptomatic peripheral vascular disease underwent examination with both conventional contrast material-enhanced arteriography and two-dimensional TOF MR angiography to evaluate the clinical usefulness of MR angiography. Postinterventional and intraoperative angiography or direct surgical exploration was the standard of reference. In 32 patients, discrepancies occurred between findings on arteriograms and those on MR angiograms; most of these discrepancies were caused by improved depiction of runoff vessels on MR angiograms. The demonstration with MR angiography of blood vessels not seen on conventional arteriograms, unidentified stenoses, or misidentified blood vessels altered surgical management in 12 patients (16%). Metal-clip artifacts obscured clinically important disease on MR angiograms in two patients. It is concluded that two-dimensional TOF MR angiography is very useful in preoperative assessment of patients with severe peripheral vascular disease.

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To assess the accuracy of magnetic resonance (MR) imaging in the evaluation of the shoulder after surgery, MR examinations were performed in 31 patients before repeated surgery, and MR findings were correlated with the subsequent operative findings. In addition, the MR findings associated with prior surgery were reviewed, including altered structure of the acromion, soft-tissue metal artifacts, a surgical trough in the humeral head, nonvisualization of the subacromial-subdeltoid fat stripe, and intermediate signal intensity within the substance of the rotator cuff on images obtained with all pulse sequences. The MR criteria for full-thickness tears in the shoulder after surgery are the presence of fluidlike signal intensity on T2-weighted images that extends through an area of the rotator cuff or the nonvisualization of a portion of the rotator cuff. With use of these criteria, six of seven full-thickness rotator cuff tears were identified at MR imaging, with an accuracy of 90%. Partial cuff tears were indistinguishable from repaired tendons. Findings at MR imaging were 74% accurate concerning the presence of impingement.

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To assess the performance of published criteria for differentiation of endometriomas from other hemorrhagic adnexal lesions with magnetic resonance (MR) imaging, 74 lesions with pathologic proof in 46 patients were evaluated by means of a 1.5-T system with use of conventional T1-weighted and either T2-weighted spin-echo (SE) or fast SE sequences. The MR images were independently presented to each of three blinded readers, and receiver operating characteristic (ROC) curve analysis was performed. The criterion of a cyst with high signal intensity on short repetition time (TR)/echo time (TE) images and shading on long TR/TE images had a mean sensitivity of 68%, mean specificity of 83%, and mean accuracy of 76% for diagnosis of endometrioma. Ancillary findings previously reported to be suggestive of endometrioma (eg, low-signal-intensity rims, adhesions, bowel tethering, and implants) were proved inaccurate and showed low interobserver agreement. ROC analysis showed similar area measurements among the three radiologists for diagnosis of endometrioma. MR imaging had only moderate accuracy in distinction of endometriomas from other hemorrhagic adnexal lesions.

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Recent improvements in magnetic resonance imaging techniques have made magnetic resonance angiography (MRA) a very useful adjunct to invasive angiography. Fifty-five limbs in 51 patients with occlusive peripheral vascular disease were studied with both MRA and contrast arteriography. The magnetic resonance and contrast arteriograms were read by radiologists and surgeons and separate interventional plans were based on each study. The MRA findings differed significantly from those of conventional arteriography in 26 limbs (48%). In every case MRA visualized all of the same vessels and hemodynamic stenoses seen on the contrast arteriogram. In 48% of the cases, however, MRA revealed additional findings. Thus the discrepancies in the two studies were always the result of the failure of the arteriogram to reveal all of the patent vessels seen on MRA. The additional information provided by MRA resulted in alteration of the interventional plan in 11 cases (22%). In nine cases (18%) target vessels suitable for use in a limb-salvage procedure were identified by MRA, although they had been missed by conventional arteriography. In all of these cases, intraoperative arteriograms confirmed the suitability of these vessels for use in technically successful bypass procedures. In two cases (4%) additional information provided by MRA identified a target runoff vessel for bypass grafting that proved to be a better alternative than the one that would have been chosen on the basis of contrast arteriography.(ABSTRACT TRUNCATED AT 250 WORDS)

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BACKGROUND: Bypass grafting to arteries of the lower leg has become standard surgical management of advanced peripheral vascular disease. Its success depends on identifying suitable distal vessels. Preoperative preparation includes imaging of the arteries of the lower leg, usually by conventional contrast arteriography. An alternative procedure, magnetic resonance (MR) angiography, has been successfully employed in patients with various cardiovascular diseases, but its possible value in patients with peripheral vascular disease has received little attention. METHODS: We used both conventional and MR angiography in preoperative studies of the lower-leg vessels of 23 patients (25 legs) with peripheral arteriosclerosis and arterial insufficiency, and developed independent therapeutic plans based on the information provided by each technique. When the plans differed, the interventional procedure judged more likely to save the limb was performed. The findings of conventional and MR angiography were verified by intraoperative arteriography, postinterventional arteriography, or direct operative exploration. RESULTS: MR angiography detected all vessels identified by conventional angiography, whereas conventional arteriography failed to detect 22 percent of the runoff vessels identified by MR angiography. The detection by MR angiography of vessels not identified by conventional angiography altered the surgical management of the disorders of four patients (17 percent) and guided successful bypass procedures. CONCLUSIONS: MR angiography is a noninvasive technique with greater sensitivity than conventional contrast arteriography for detecting distal runoff vessels in patients with peripheral arterial occlusive disease.

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Fifty-four independent scans were performed in two volunteers covering one anatomic region in each (the brain and knee) with the purpose of ascertaining the agreement between predicted and measured signal-to-noise ratios (SNR). Systematically varied parameters were number of excitations (NEX), field of view (FOV), section thickness (dz), and the number of phase-encoding steps (Ny). Correlation coefficients of measured versus predicted SNR were 0.82 and 0.86, respectively, in the anatomies studied. Significantly improved correlations were found for data subpopulations in which NEX was held constant. To assess the criteria guiding reader preference, a blinded study was performed in which radiologists were asked to rate images from least to most desirable. In order to quantitatively determine the criteria for reader preference, plots of mean rating versus SNR, voxel volume, and an image quality index [IQI = SNR/(voxel volume)] were performed. The latter was found to be a better predictor of reader preference than either SNR or spatial resolution alone. The data suggests T1-weighted scan protocols yielding SNR of approximately 20 are preferable with any excess SNR being traded for smaller voxel size or shorter scan times.

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The caloric expenditure of 44 healthy, lean and obese women, 8 of whom were trained athletes, was measured by indirect calorimetry. Body composition was determined. Ages ranged from 18-65 yr and body weights from 43-143 kg. Stepwise, multiple-regression analysis was used to determine whether one or several variables best predicted the resting metabolic rate (RMR) of the women. The RMR and the thermic effect of food (TEF) were measured before and after the women consumed a mixed breakfast meal. The results showed that the currently available tables and regression equations overestimate the RMR of healthy women by 7-14%. Body weight was highly related to the RMR, and stepwise inclusions of various variables did not improve predictions of RMR. The slopes of the regression lines for nonathletes and athletes were significantly different. Regression equations for predicting RMR of women were developed: Nonathletes RMR = 795 + 7.18 kg WT; Athletes RMR = 50.4 + 21.1 kg WT. The range of RMR per kilogram body weight was wide for nonathletic, but narrow for athletic women. The metabolism of some lean and obese, nonathletic women was highly efficient, predisposing these women for developing and maintaining body fat. The TEFs were indistinguishable between nonathletic and athletic women, and formed a continuum from the lightest to the heaviest woman.

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Hepatic cirrhosis with portal hypertension and gastroesophageal hemorrhage is a disease complex that continues to be treated by surgical portasystemic shunts. Whether or not a reduction or diversion of portal blood flow to the liver adversely affects the ability of the liver to maintain fuel homeostasis via gluconeogenesis, glycogenolysis, and ketogenesis is unknown. 11 patients with biopsy-proven severe hepatic cirrhosis were studied before and after distal splenorenal or mesocaval shunts. Hepatic, portal, and renal blood flow rates and glucose, lactate, pyruvate, glycerol, amino acids, ketone bodies, free fatty acids, and triglyceride arteriovenous concentration differences were determined to calculate net precursor-product exchange rates across the liver, gut, and kidney. The study showed that hepatic contribution of glucose and ketone bodies and the caloric equivalents of these fuels delivered to the blood was not adversely affected by either a distal splenorenal or mesocaval shunt. In addition to these general observations, isolated findings emerged. Mesocaval shunts reversed portal venous blood and functionally converted this venous avenue into hepatic venous blood. The ability of the kidney to make a substantial net contribution of ketone bodies to the blood was also observed.

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