Long-term labeling and imaging of synaptically connected neuronal networks in vivo using double-deletion-mutant rabies viruses.

Rabies-virus-based monosynaptic tracing is a widely used technique for mapping neural circuitry, but its cytotoxicity has confined it primarily to anatomical applications. Here we present a second-generation system for labeling direct inputs to targeted neuronal populations with minimal toxicity, using double-deletion-mutant rabies viruses. Viral spread requires expression of both deleted viral genes in trans in postsynaptic source cells. Suppressing this expression with doxycycline following an initial period of viral replication reduces toxicity to postsynaptic cells. Longitudinal two-photon imaging in vivo indicated that over 90% of both presynaptic and source cells survived for the full 12-week course of imaging. Ex vivo whole-cell recordings at 5 weeks postinfection showed that the second-generation system perturbs input and source cells much less than the first-generation system. Finally, two-photon calcium imaging of labeled networks of visual cortex neurons showed that their visual response properties appeared normal for 10 weeks, the longest we followed them.

Third-generation rabies viral vectors allow nontoxic retrograde targeting of projection neurons with greatly increased efficiency.

Rabies viral vectors have become important components of the systems neuroscience toolkit, allowing both direct retrograde targeting of projection neurons and monosynaptic tracing of inputs to defined postsynaptic populations, but the rapid cytotoxicity of first-generation (ΔG) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant (ΔGL) rabies viral vectors, showing that they efficiently retrogradely infect projection neurons and express recombinases effectively but with little to no detectable toxicity; more recently, we have shown that ΔGL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here, we introduce third-generation (ΔL) rabies viral vectors, which appear to be as nontoxic as second-generation ones but have the major advantage of growing to much higher titers, resulting in significantly increased numbers of retrogradely labeled neurons in vivo.

"Self-inactivating" rabies viruses are susceptible to loss of their intended attenuating modification.

Monosynaptic tracing using rabies virus is an important technique in neuroscience, allowing brain-wide labeling of neurons directly presynaptic to a targeted neuronal population. A 2017 article reported the development of a noncytotoxic version-a major advance-based on attenuating the rabies virus by the addition of a destabilization domain to the C terminus of a viral protein. However, this modification did not appear to hinder the ability of the virus to spread between neurons. We analyzed two viruses provided by the authors and show here that both were mutants that had lost the intended modification, explaining the paper's paradoxical results. We then made a virus that actually did have the intended modification in at least the majority of virions and found that it did not spread efficiently under the conditions described in the original paper, namely, without an exogenous protease being expressed in order to remove the destabilization domain. We found that it did spread when the protease was supplied, although this also appeared to result in the deaths of most source cells by 3 wk postinjection. We conclude that the new approach is not robust but that it could become a viable technique given further optimization and validation.

Adjustable intragastric balloon for treatment of obesity: a multicentre, open-label, randomised clinical trial.

BACKGROUND: Intragastric balloons are anatomy-preserving, minimally invasive obesity therapies. Enhanced tolerance and durability could help broaden clinical adoption. We investigated the safety and efficacy of an adjustable intragastric balloon (aIGB) in adults with obesity. METHODS: In this prospective, multicentre, open-label, randomised clinical trial done at seven US sites, adults aged 22-65 years with obesity were randomly assigned (2:1) to aIGB with lifestyle intervention or lifestyle intervention alone (control) for 32 weeks. Balloon volume could be increased to facilitate weight loss or decreased for tolerability. Coprimary endpoints included mean percentage total bodyweight loss and responder rate (≥5% total bodyweight loss) at 32 weeks. We used a multiple imputed intention-to-treat population analysis. This study was registered with ClinicalTrials.gov, NCT02812160. FINDINGS: Between Aug 9, 2016, and Dec 7, 2018, we randomly assigned 288 patients to aIGB (n=187 [65%]) or control (n=101 [35%]) groups. Mean total bodyweight loss at 32 weeks was 15·0% (95% CI 13·9-16·1) in the aIGB group versus 3·3% (2·0-4·6) in the control group (p<0·0001). Clinical response was observed in 171 (92%) patients in the aIGB group. Adjustments to the aIGB occurred in 145 (80%) patients for weight loss plateau or intolerance. Upward volume adjustment facilitated an additional mean 5·2% (4·5-5·8) total bodyweight loss. Downward volume adjustment allowed 21 (75%) patients in the aIGB group to complete the full duration of therapy. Intolerance caused early removal of the device in 31 (17%) patients. No micronutrient deficiencies were observed in the aIGB cohort. Device-related serious adverse events were observed in seven (4%) patients, without any deaths. INTERPRETATION: When aIGB was combined with lifestyle modification, significant weight loss was achieved and maintained for 6 months following removal. Balloon volume adjustability permitted individualised therapy, maximising weight loss and tolerance. FUNDING: Spatz Medical.

Development and validation of blood-based prognostic biomarkers for severity of COVID disease outcome using EpiSwitch 3D genomic regulatory immuno-genetic profiling.

The COVID-19 pandemic has raised several global public health challenges to which the international medical community have responded. Diagnostic testing and the development of vaccines against the SARS-CoV-2 virus have made remarkable progress to date. As the population is now faced with the complex lifestyle and medical decisions that come with living in a pandemic, a forward-looking understanding of how a COVID-19 diagnosis may affect the health of an individual represents a pressing need. Previously we used whole genome microarray to identify 200 3D genomic marker leads that could predict mild or severe COVID-19 disease outcomes from blood samples in a multinational cohort of COVID-19 patients. Here, we focus on the development and validation of a qPCR assay to accurately predict severe COVID-19 disease requiring intensive care unit (ICU) support and/or mechanical ventilation. From 200 original biomarker leads we established a classification model containing six markers. The markers were qualified and validated on 38 COVID-19 patients from an independent cohort. Overall, the six-marker model obtained a positive predictive value of 93% and balanced accuracy of 88% across 116 patients for the prognosis of COVID-19 severity requiring ICU care/ventilation support. The six-marker signature identifies individuals at the highest risk of developing severe complications in COVID-19 with high predictive accuracy and can assist in patient prognosis and clinical management decisions.

Monosynaptic Tracing Success Depends Critically on Helper Virus Concentrations.

Monosynaptically-restricted transsynaptic tracing using deletion-mutant rabies virus (RV) has become a widely used technique in neuroscience, allowing identification, imaging, and manipulation of neurons directly presynaptic to a starting neuronal population. Its most common implementation is to use Cre mouse lines in combination with Cre-dependent "helper" adeno-associated viral vectors (AAVs) to supply the required genes to the targeted population before subsequent injection of a first-generation (ΔG) rabies viral vector. Here we show that the efficiency of transsynaptic spread and the degree of nonspecific labeling in wild-type control animals depend strongly on the concentrations of these helper AAVs. Our results suggest practical guidelines for achieving good results.

Improving Hemostasis in Sleeve Gastrectomy With Alternative Stapler.

BACKGROUND: Staple line bleeding can be a major intra-operative complication during laparoscopic sleeve gastrectomy, requiring reinforcing interventions that may diminish the integrity of the staple line and put patients at risk for postoperative hemorrhage or leak. To improve outcomes associated with surgery, surgeons may benefit from an alternative stapler that produces a drier staple line and requires less staple line manipulation. METHODS: Sixty consecutive laparoscopic sleeve gastrectomy procedures were performed by three surgeons; 30 sleeves using the AEON™ Endostapler on THICK MODE and 30 using the Echelon Flex™ Powered Stapler with pulse technique. Stapler performance was measured by incidence and degree of staple line bleeding. Images of the first firing and fundus were taken with the laparoscope 10 seconds after the final firing. Images were evaluated by a third-party blinded evaluator and given a "bleeding score," a qualitative measure of intra-operative staple-line bleeding (1 = no bleeding to 5 = profuse bleeding). RESULTS: The AEON™ Endostapler demonstrated a lower mean (± standard error) "bleeding score" versus the Echelon Flex™ (2.1 ± 0.1 vs. 2.6 ± 0.1; p = 0.01). The AEON™ Endostapler had 15 cases (50%) with no bleeding at the fundus; the Echelon Flex™ had 7 cases (23%) with no bleeding at the fundus. The AEON™ Endostapler had 0 cases (0%) with profuse bleeding; the Echelon Flex™ had 2 cases (7%) with profuse bleeding. CONCLUSION: The AEON™ Endostapler is a significantly drier alternative to the Echelon Flex™ Powered Stapler, producing a much drier staple line and decreasing the need for other bleeding control methods.

Monosynaptic tracing: a step-by-step protocol.

Monosynaptic tracing using deletion-mutant rabies virus allows whole-brain mapping of neurons that are directly presynaptic to a targeted population of neurons. The most common and robust way of implementing it is to use Cre mouse lines in combination with Cre-dependent adeno-associated viral vectors for expression of the required genes in the targeted neurons before subsequent injection of the rabies virus. Here we present a step-by-step protocol for performing such experiments using first-generation (ΔG) rabies viral vectors.

Nontoxic, double-deletion-mutant rabies viral vectors for retrograde targeting of projection neurons.

Recombinant rabies viral vectors have proven useful for applications including retrograde targeting of projection neurons and monosynaptic tracing, but their cytotoxicity has limited their use to short-term experiments. Here we introduce a new class of double-deletion-mutant rabies viral vectors that left transduced cells alive and healthy indefinitely. Deletion of the viral polymerase gene abolished cytotoxicity and reduced transgene expression to trace levels but left vectors still able to retrogradely infect projection neurons and express recombinases, allowing downstream expression of other transgene products such as fluorophores and calcium indicators. The morphology of retrogradely targeted cells appeared unperturbed at 1 year postinjection. Whole-cell patch-clamp recordings showed no physiological abnormalities at 8 weeks. Longitudinal two-photon structural and functional imaging in vivo, tracking thousands of individual neurons for up to 4 months, showed that transduced neurons did not die but retained stable visual response properties even at the longest time points imaged.

Nonsurgical Facial Rejuvenation Procedures in Patients Under 50 Prior to Undergoing Facelift: Habits, Costs, and Results.

Background: Facial rejuvenation in patients younger than 50 years of age has experienced an unprecedented growth with multimodality nonsurgical and less invasive rhytidectomy techniques. Objectives: To analyze the nonsurgical treatment habits of patients prior to undergoing rhytidectomy at <50 years of age. Methods: Retrospective study to enlist patients who underwent primary rhytidectomy at age <50 years between January 1, 2003 and December 31, 2013 by the senior author (AAJ) to complete a survey. Results: One hundred and fifty-seven patients were surveyed. Patients had nonsurgical rejuvenation starting at an average age of 37 years and rhytidectomy at an average age of 44 years. Thirty-two percent of responders had injectable treatments prior to their facelift, reporting a mean of 7 rounds of injectable treatments prior to pursuing rhytidectomy. Sixteen percent of responders had laser skin resurfacing undergoing 4 separate treatments prior to rhytidectomy, and 10% had energy-based facial tightening treatments one time prior to their rhytidectomy. Average expenditure on nonsurgical treatments prior to rhytidectomy was $7000 cumulatively. Fifty-nine percent of patients who went on to rhytidectomy did not report regret over this cost expenditure. Patients reported that they appeared 4 years younger after nonsurgical intervention, and 8 years younger after their facelift, a statistically significant difference (P = .048). Conclusions: Patients undergoing rhytidectomy <50 years old begin less invasive facial rejuvenation treatments at an even earlier age. The majority of these patients did not regret the costs associated with noninvasive treatments, even though they saw that rhytidectomy provided a greater rejuvenation effect. Rhytidectomy surgeons should incorporate nonsurgical techniques into their practice to best serve the needs of the modern aging face patient.

Randomized sham-controlled trial evaluating efficacy and safety of endoscopic gastric plication for primary obesity: The ESSENTIAL trial.

OBJECTIVE: Evaluate safety and efficacy of the pose™ procedure for obesity treatment. METHODS: Subjects with Class I to II obesity were randomized (2:1) to receive active or sham procedure, after each investigator performed unblinded lead-in cases. All subjects were provided low-intensity lifestyle therapy. Efficacy end points were the mean difference in percent total body weight loss (%TBWL) at 12 months between randomized groups, and responder rate achieving ≥5% TBWL. The primary safety end point was incidence of reported adverse events. RESULTS: Three hundred thirty-two subjects were randomized (active, n = 221; sham, n = 111); thirty-four subjects were included in the unblinded lead-in cohort. Twelve-month results were mean TBWL 7.0 ± 7.4% in lead-in, 4.95 ± 7.04% in active, and 1.38 ± 5.58% in sham groups, respectively. Responder rate was 41.55% in active and 22.11% in sham groups, respectively (P < 0.0001); mean responder result was 11.5% TBWL. The differences observed between active and sham groups for co-primary end points were statistically significant (P < 0.0001); however, super superiority margin as set forth in the study design was not met. No unanticipated adverse events or deaths occurred. Procedure-related serious adverse event rates were 5.0% (active) and 0.9% (sham), P = 0.068. CONCLUSIONS: The pose procedure was safe and resulted in statistically significant and clinically meaningful weight loss over sham through 1 year.

Comparing rates of distal edge necrosis in deep-plane vs subcutaneous cervicofacial rotation-advancement flaps for facial cutaneous Mohs defects.

IMPORTANCE: The cervicofacial rotation-advancement flap is commonly used for facial defects. Decreasing the rate of distal edge necrosis (DEN) encountered with this flap would help prevent complications in sensitive areas such as the eyelid, lip, and nose. OBJECTIVE: To compare the untoward occurrence of DEN between 2 surgical dissection methods for reconstructive cervicofacial rotation-advancement flaps. DESIGN, SETTING, PARTICIPANTS, AND EXPOSURE: A review was conducted of 88 patients who underwent cervicofacial flap reconstruction for Mohs ablative surgery between January 1, 2003, and June 30, 2012, by the senior author (A.A.J.). All patients had periorbital, midfacial, cervical, and/or lateral temporal/forehead defects following Mohs surgical ablation. Patients were categorized into 1 of 2 groups on the basis of the surgical technique used: subcutaneous (SC) cervicofacial elevation or deep-plane (DP) cervicofacial elevation. Subcategories of smokers and nonsmokers within each group were further reviewed. Statistical analysis of DEN between categories and subcategories was performed. RESULTS: Sixty-nine patients were in the SC group and 19 were in the DP group. The mean defect size among both groups was 14.3 cm(2). The rate of active or recent smokers was 23% in the SC group and 11% in the DP group. The rate of DEN among nonsmokers in the SC group was 23% (n = 53) compared with 0% in the 17 DP nonsmokers (P = .03). The rate of smokers with DEN in the SC group was 75% and 0% in the DP group (P = .09). The mean area of DEN in the SC group was 0.8 cm(2). CONCLUSIONS AND RELEVANCE: Our statistically significant data indicate that DP dissection is a superior technique for avoiding DEN in nonsmokers. We found better outcomes in smokers as well. Thus, we strongly advocate the use of the DP approach as the criterion standard in cervicofacial flap elevation. LEVEL OF EVIDENCE: 3.

Action of topical thyroid hormone analogue, triiodothyroacetic acid in reversing glucocorticoid-induced skin atrophy in humans.

The present study concerns the effect of topical treatment with a cream formulation of triiodothyroacetic acid (TRIAC) in comparison with a placebo preparation in producing a reversal of skin atrophy induced by long-term employment of topical glucocorticoid therapy in humans. A total of 39 patients with clinically verified skin atrophy due to long-term use of topical potent glucocorticoids were randomized. The changes in skin thickness, elastic fibers, and hyaluronic acid were evaluated by means of sonography and histology. After 8 weeks' treatment, the skin thickness measured by sonography increased by 16% in the epidermis, 8% in the dermis, and epidermis + dermis in the placebo group. In the TRIAC 0.1% group, the corresponding values were 24% ( p=0.063) in the epidermis, 28% ( p=0.042) in the dermis, and 25% ( p=0.039) in the epidermis + dermis. After 8 weeks, in the placebo group, the skin thickness measured by biopsy increased by 5% in the epidermis, epidermis + dermis, and 6% in the dermis. In the TRIAC 0.1% group, the corresponding values were 31% ( p=0.041) in the epidermis, 46% ( p=0.041) in the dermis and 44% ( p=0.043) in the epidermis + dermis. After 8 weeks, the elastic fibers of moderately irregular and thickened fibers increased by 56% in the placebo group and 100% ( p=0.043) in the TRIAC 0.1 group. This study indicates that topical treatment with TRIAC appears to reverse glucocorticoid-induced skin atrophy under the narrow conditions tested.

Neural substrates of emotion as revealed by functional magnetic resonance imaging.

OBJECTIVES: To examine the brain circuitry involved in emotional experience and determine whether the cerebral hemispheres are specialized for positive and negative emotional experience. BACKGROUND: Recent research has provided a preliminary sketch of the neurologic underpinnings of emotional processing involving specialized contributions of limbic and cortical brain regions. Electrophysiologic, functional imaging, and Wada test data have suggested positive, approach-related emotions are associated with left cerebral hemisphere regions, whereas negative, withdrawal-related emotions appear to be more aligned with right hemisphere mechanisms. METHOD: These emotional-neural associations were investigated using functional magnetic resonance imaging in 10 healthy controls with 20 positively and 20 negatively valenced pictures from the International Affective Picture System in a counterbalanced order. Pictures were viewed within a 1.5 Telsa scanner through computerized video goggles. RESULTS: Emotional pictures resulted in significantly increased blood flow bilaterally in the mesial frontal lobe/anterior cingulate gyrus, dorsolateral frontal lobe, amygdala/anterior temporal regions, and cerebellum. Negative emotional pictures resulted in greater activation of the right hemisphere, and positive pictures caused greater activation of the left hemisphere. CONCLUSIONS: Results are consistent with theories emphasizing the importance of circuitry linking subcortical structures with mesial temporal, anterior cingulate, and frontal lobe regions in emotion and with the valence model of emotion that posits lateralized cerebral specialization for positive and negative emotional experience.

Functional MR imaging study of language-related differences in bilingual cerebellar activation.

BACKGROUND AND PURPOSE: Reports in the monolingual literature suggest that the cerebellum has an important role in language processing. The purpose of this study was to determine whether bilingual cerebellar functional MR imaging (fMRI) activation differs during the performance of comparable tasks in subjects' primary and secondary languages. METHODS: Eight bilingual, right-handed individuals underwent echo-planar fMRI at 1.5 T. They performed semantic (noun-verb association) and phonological (rhyming) tasks in Spanish (primary language) and English (secondary language). Individual and group functional datasets were analyzed using Statistical Parametric Mapping software (SPM99; P <.001 with a 10-voxel spatial extent threshold) and overlaid on T1-weighted anatomic images normalized to a standard (Montreal Neurologic Institute) space. Analysis of variance was performed on laterality indices derived from voxel counts in cerebellar regions of interest (ROIs). Subtraction of group-averaged normalized results from the combined Spanish tasks from the combined English tasks was also performed within SPM99 (P <.001 activation threshold). RESULTS: Significantly greater lateralilty indices were noted in the English tasks than in the Spanish tasks (mean Spanish LI, 0.3286; mean English LI, 0.5141 [P =.0143]). Overall, more robust activation was seen in the English tasks than in the Spanish tasks. Areas of significantly greater activation existed in the English tasks as compared with the Spanish tasks; these areas were more prominent in the left cerebellar hemisphere. CONCLUSION: Although both English and Spanish language tasks demonstrate left cerebellar dominance, English tasks demonstrate greater left hemispheric lateralization.

Dose-response effects of tri-iodothyroacetic acid (Triac) and other thyroid hormone analogues on glucocorticoid-induced skin atrophy in the haired mouse.

Thyroid hormones have an influence on the connective tissue biology of the skin and, theoretically, topically applied thyroid hormones or hormone analogues could have a stimulatory effect on collagen synthesis. In this investigation the effect of topical tri-iodothyroacetic acid (Triac) and other thyroid hormone analogues were tested for their effect in preventing betamethasone-induced skin atrophy in the normal haired mouse. Triac, tri-iodoproprionic acid (Triprop) and the synthetically developed thyroid hormone analogue KB-026 and 2 different Triac cream formulations were applied along with betamethasone on shaved mouse skin. Triac in daily doses of 1 nmol/cm2 and higher was able to block the betamethasone-induced skin atrophy in mice skin. In high doses, Triprop and KB-026 also had a blocking effect. Triac alone had a stimulatory effect on dermal thickness. This study indicates that thyroid hormone analogues may be used to prevent corticosteroid-induced skin atrophy.