Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes—such as transcriptional profiles—at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays. Keywords: single-cell RNA-seq; pooled screen; CRISPR; epistasis; genetic interaction

Allergen recognition by specific effector Th2 cells enables IL-2-dependent activation of regulatory T cell responses in humans

medRxiv preprintType 2 allergen-specific T cells are essential for the induction and maintenance of allergies to foods, and Tregs specific for these allergens are assumed to be involved in their resolution. However, it has not been convincingly demonstrated whether allergen-specific Treg responses are responsible for the generation of oral tolerance in humans. We observed that sustained food allergen exposure in the form of oral immunotherapy resulted in increased frequency of Tregs only in individuals with lasting clinical tolerance. We sought to identify regulatory components of the CD4+ T-cell response to food allergens by studying their functional activation over time in vitro and in vivo. Two subsets of Tregs expressing CD137 or CD25/OX40 were identified with a delayed kinetics of activation compared with clonally enriched pathogenic effector Th2 cells. Treg activation was dependent on IL-2 derived from effector T cells. In vivo exposure to peanut in the form of an oral food challenge of allergic subjects induced a delayed and persistent activation of Tregs after initiation of the allergen-specific Th2 response. The novel finding of our work is that a sustained wave of Treg activation is induced by the release of IL-2 from Th2 effector cells, with the implication that therapeutic administration of IL-2 could improve current OIT approaches.We thank A. Grishin and M. Masilamani for their leadership of laboratory studies of CoFAR7. We thank the staff of the clinical research units at each participating center, the Statistical and Clinical Coordinating Center, and K. Peyton, the SACCC Project Manager. We thank J. Poyser, Project Manager for CoFAR7 Program (National Institutes of Health [NIH]/National Institute of Allergy and Infectious Diseases [NIAID]). Finally, we thank the families who kindly participated.Peer reviewe

Understanding acute ankle ligamentous sprain injury in sports

This paper summarizes the current understanding on acute ankle sprain injury, which is the most common acute sport trauma, accounting for about 14% of all sport-related injuries. Among, 80% are ligamentous sprains caused by explosive inversion or supination. The injury motion often happens at the subtalar joint and tears the anterior talofibular ligament (ATFL) which possesses the lowest ultimate load among the lateral ligaments at the ankle. For extrinsic risk factors to ankle sprain injury, prescribing orthosis decreases the risk while increased exercise intensity in soccer raises the risk. For intrinsic factors, a foot size with increased width, an increased ankle eversion to inversion strength, plantarflexion strength and ratio between dorsiflexion and plantarflexion strength, and limb dominance could increase the ankle sprain injury risk. Players with a previous sprain history, players wearing shoes with air cells, players who do not stretch before exercising, players with inferior single leg balance, and overweight players are 4.9, 4.3, 2.6, 2.4 and 3.9 times more likely to sustain an ankle sprain injury. The aetiology of most ankle sprain injuries is incorrect foot positioning at landing – a medially-deviated vertical ground reaction force causes an explosive supination or inversion moment at the subtalar joint in a short time (about 50 ms). Another aetiology is the delayed reaction time of the peroneal muscles at the lateral aspect of the ankle (60–90 ms). The failure supination or inversion torque is about 41–45 Nm to cause ligamentous rupture in simulated spraining tests on cadaver. A previous case report revealed that the ankle joint reached 48 degrees inversion and 10 degrees internal rotation during an accidental grade I ankle ligamentous sprain injury during a dynamic cutting trial in laboratory. Diagnosis techniques and grading systems vary, but the management of ankle ligamentous sprain injury is mainly conservative. Immobilization should not be used as it results in joint stiffness, muscle atrophy and loss of proprioception. Traditional Chinese medicine such as herbs, massage and acupuncture were well applied in China in managing sports injuries, and was reported to be effective in relieving pain, reducing swelling and edema, and restoring normal ankle function. Finally, the best practice of sports medicine would be to prevent the injury. Different previous approaches, including designing prophylactice devices, introducing functional interventions, as well as change of games rules were highlighted. This paper allows the readers to catch up with the previous researches on ankle sprain injury, and facilitate the future research idea on sport-related ankle sprain injury

Surgical Management of Lumbosacral Plexus Tumors

Lumbosacral plexus tumors are uncommon, and because of their deep location and proximity to critical nerves subserving lower extremity function, understanding surgical approaches and short-term outcomes is important. In a retrospective case series of lumbosacral plexus tumor surgeries performed from May 2000 to July 2021 by a single neurosurgeon, demographic information, clinical presentation, imaging studies, and operative outcomes were analyzed. A total of 42 patients with mean age of 48.3 years (range, 16–84 years) underwent surgery for a lumbosacral plexus tumor. Patients presented with leg pain (n = 25; 59.5%), followed by back/flank pain (n = 5; 11.9%), abdominal/pelvic pain (n = 5; 11.9%), leg weakness (n = 5; 11.9%), and leg numbness (n = 3; 7.1%). The most common tumor pathology was schwannoma (n = 20; 50.0%) followed by neurofibroma (n = 9; 22.5%). A retroperitoneal approach was used in all cases. Gross total resection was achieved in 23 (54.8%) patients, and only 1 (2.4%) patient exhibited symptomatic tumor recurrence after subtotal resection of a malignant tumor. Mean follow-up was 33.1 months (range, 1–96 months). Postoperatively, patient neurological status remained unchanged or improved (n = 37; 88.1%). Complications were infrequent, with 4 (9.5%) patients experiencing new sensory symptoms and 1 patient (2.4%) experiencing new anticipated motor weakness after en bloc resection of a malignant tumor. Indications for surgery include pain and/or neurological symptoms attributable to the lesion or large size if asymptomatic. Careful study of preoperative imaging is necessary to determine the best approach. Intraoperative nerve stimulation is essential to preserve function and guide extent of resection in benign tumors

Massively-parallel single nucleus RNA-seq with DroNc-seq

Single nucleus RNA-seq (sNuc-seq) profiles RNA from tissues that are preserved or cannot be dissociated, but does not provide the throughput required to analyse many cells from complex tissues. Here, we develop DroNc-seq, massively parallel sNuc-Seq with droplet technology. We profile 39,111 nuclei from mouse and human archived brain samples to demonstrate sensitive, efficient and unbiased classification of cell types, paving the way for systematic charting of cell atlases

Massively parallel single-nucleus RNA-seq with DroNc-seq.

Single-nucleus RNA sequencing (sNuc-seq) profiles RNA from tissues that are preserved or cannot be dissociated, but it does not provide high throughput. Here, we develop DroNc-seq: massively parallel sNuc-seq with droplet technology. We profile 39,111 nuclei from mouse and human archived brain samples to demonstrate sensitive, efficient, and unbiased classification of cell types, paving the way for systematic charting of cell atlases