Massively parallel disruption of enhancers active in human neural stem cells.

Changes in gene regulation have been linked to the expansion of the human cerebral cortex and to neurodevelopmental disorders, potentially by altering neural progenitor proliferation. However, the effects of genetic variation within regulatory elements on neural progenitors remain obscure. We use sgRNA-Cas9 screens in human neural stem cells (hNSCs) to disrupt 10,674 genes and 26,385 conserved regions in 2,227 enhancers active in the developing human cortex and determine effects on proliferation. Genes with proliferation phenotypes are associated with neurodevelopmental disorders and show biased expression in specific fetal human brain neural progenitor populations. Although enhancer disruptions overall have weaker effects than gene disruptions, we identify enhancer disruptions that severely alter hNSC self-renewal. Disruptions in human accelerated regions, implicated in human brain evolution, also alter proliferation. Integrating proliferation phenotypes with chromatin interactions reveals regulatory relationships between enhancers and their target genes contributing to neurogenesis and potentially to human cortical evolution.

Piloting delivery of PfSPZ vaccines for malaria through a cryogenic vaccine cold chain to travel and military medicine clinics.

BACKGROUND: PfSPZ vaccines comprising Plasmodium falciparum (Pf) sporozoites (SPZ) have demonstrated > 90% protection against variant Pf malaria infections for at least 12 weeks; they are the only vaccines with the level of efficacy necessary to protect travellers. PfSPZ are eukaryotic cells stabilized by cryopreservation and distributed using a cryogenic (below -150 °C) cold chain. The Ebola vaccine and mRNA vaccines against SARS-CoV-2 pioneered uptake of vaccines requiring non-standard ultra-low temperature cold chains. The cryogenic cold chain using liquid nitrogen (LN2) vapour phase (LNVP) cryoshippers, is simpler, more efficient than -80, -20 or 2-8 °C cold chains, and does not use electricity. This study was conducted to evaluate implementation and integration of a cryogenically distributed vaccine at travel and military immunization clinics. METHODS: We conducted sequential 28-day studies evaluating vaccine shipping, storage, maintenance and accession at two US military and two civilian travel health/immunization clinics. In each clinic, personnel were trained in equipment use, procurement and handling of LN2, temperature monitoring and inventory record keeping by in-person or video instruction. RESULTS: Sites required 2-4 h/person for two persons to assimilate and develop the expertise to manage vaccine storage and LNVP operations. LN2 for recharging cryoshippers was delivered every 1-2 weeks. Vaccine ordering, receipt, storage and inventory control was conducted effectively. Simulated single dose vaccine cryovial retrieval and thawing were performed successfully in different travel clinic settings. Continuous temperature monitoring at each site was maintained with only one short excursion above -150 °C (-145 °C) through shipping, use and reverse logistics. Staff, during and at study conclusion, provided feedback that has been incorporated into our models for cold chain logistics. CONCLUSIONS: These studies demonstrated that the training in delivery, storage, administration and integration of PfSPZ vaccines can be successfully managed in different immunization clinic settings for travellers and military personnel.

Can Perioperative Antibiotic Choice Impact Rates of Infectious Complications After Percutaneous Nephrolithotomy? A Single-Blind, Prospective Randomized Trial.

Objective: National guidelines recommend periprocedural antibiotics before percutaneous nephrolithotomy (PCNL), yet it is not clear which is superior. We conducted a randomized trial to compare two guideline-recommended antibiotics: ciprofloxacin (cipro) vs cefazolin, on PCNL outcomes, focusing on the development of systemic inflammatory response syndrome (SIRS) criteria. Methods: Adult patients who were not considered high risk for surgical or infectious complications and undergoing PCNL were randomized to receive either cipro or cefazolin perioperatively. All had negative preoperative urine cultures. Demographic and perioperative data were collected, including SIRS criteria, intraoperative urine culture, duration of hospitalization, and need for intensive care. SIRS is defined by ≥2 of the following: body temperature <96.8°F or >100.4°F, heart rate >90 bpm, respiratory rate >20 per minute, and white blood cell count <4000 or >12,000 cells/mm3. Results: One hundred forty-seven patients were enrolled and randomized (79 cefazolin and 68 cipro). All preoperative characteristics were similar (p > 0.05), except for mean age, which was higher in the cipro group (64 vs 57 years, p = 0.03). Intra- and postoperative findings were similar, with no difference between groups (p > 0.05), except a longer mean hospital stay in the cefazolin group (2 hours longer, p = 0.02). There was no difference between SIRS episodes in both univariate and multivariate analyses. Conclusions: Despite the relatively broader coverage for urinary tract pathogens with ciprofloxacin, this prospective randomized trial did not show superiority over cefazolin. Our findings therefore support two appropriate options for perioperative antibiotic prophylaxis in patients undergoing PCNL who are nonhigh risk for infectious complications.

The functional and evolutionary impacts of human-specific deletions in conserved elements.

Conserved genomic sequences disrupted in humans may underlie uniquely human phenotypic traits. We identified and characterized 10,032 human-specific conserved deletions (hCONDELs). These short (average 2.56 base pairs) deletions are enriched for human brain functions across genetic, epigenomic, and transcriptomic datasets. Using massively parallel reporter assays in six cell types, we discovered 800 hCONDELs conferring significant differences in regulatory activity, half of which enhance rather than disrupt regulatory function. We highlight several hCONDELs with putative human-specific effects on brain development, including HDAC5, CPEB4, and PPP2CA. Reverting an hCONDEL to the ancestral sequence alters the expression of LOXL2 and developmental genes involved in myelination and synaptic function. Our data provide a rich resource to investigate the evolutionary mechanisms driving new traits in humans and other species.

The Erector Spinae Plane Block as Novel Therapy for Renal Colic: A Case Series.

We present a 10-patient case series supporting the use of the erector spinae plane block (ESPB) as a novel approach for the treatment of acute pain from renal colic. An in-plane needle approach was used with either transverse or longitudinal orientation of the ultrasound probe on the affected side, in either seated or prone patient position. These cases showed significant improvement in patient reported pain; suggesting that the ESPB can be used safely and effectively for either primary or adjunctive treatment of acute pain due to renal colic in the emergency department.

4-Aminopyridine Induces Nerve Growth Factor to Improve Skin Wound Healing and Tissue Regeneration.

The discovery of ways to enhance skin wound healing is of great importance due to the frequency of skin lesions. We discovered that 4-aminopyridine (4-AP), a potassium channel blocker approved by the FDA for improving walking ability in multiple sclerosis, greatly enhances skin wound healing. Benefits included faster wound closure, restoration of normal-appearing skin architecture, and reinnervation. Hair follicle neogenesis within the healed wounds was increased, both histologically and by analysis of K15 and K17 expression. 4-AP increased levels of vimentin (fibroblasts) and alpha-smooth muscle actin (α-SMA, collagen-producing myofibroblasts) in the healed dermis. 4-AP also increased neuronal regeneration with increased numbers of axons and S100+ Schwann cells (SCs), and increased expression of SRY-Box Transcription Factor 10 (SOX10). Treatment also increased levels of transforming growth factor-ß (TGF-ß), substance P, and nerve growth factor (NGF), important promoters of wound healing. In vitro studies demonstrated that 4-AP induced nerve growth factor and enhanced proliferation and migration of human keratinocytes. Thus, 4-AP enhanced many of the key attributes of successful wound healing and offers a promising new approach to enhance skin wound healing and tissue regeneration.

Acute axon damage and demyelination are mitigated by 4-aminopyridine (4-AP) therapy after experimental traumatic brain injury.

Damage to long axons in white matter tracts is a major pathology in closed head traumatic brain injury (TBI). Acute TBI treatments are needed that protect against axon damage and promote recovery of axon function to prevent long term symptoms and neurodegeneration. Our prior characterization of axon damage and demyelination after TBI led us to examine repurposing of 4-aminopyridine (4-AP), an FDA-approved inhibitor of voltage-gated potassium (Kv) channels. 4-AP is currently indicated to provide symptomatic relief for patients with chronic stage multiple sclerosis, which involves axon damage and demyelination. We tested clinically relevant dosage of 4-AP as an acute treatment for experimental TBI and found multiple benefits in corpus callosum axons. This randomized, controlled pre-clinical study focused on the first week after TBI, when axons are particularly vulnerable. 4-AP treatment initiated one day post-injury dramatically reduced axon damage detected by intra-axonal fluorescence accumulations in Thy1-YFP mice of both sexes. Detailed electron microscopy in C57BL/6 mice showed that 4-AP reduced pathological features of mitochondrial swelling, cytoskeletal disruption, and demyelination at 7 days post-injury. Furthermore, 4-AP improved the molecular organization of axon nodal regions by restoring disrupted paranode domains and reducing Kv1.2 channel dispersion. 4-AP treatment did not resolve deficits in action potential conduction across the corpus callosum, based on ex vivo electrophysiological recordings at 7 days post-TBI. Thus, this first study of 4-AP effects on axon damage in the acute period demonstrates a significant decrease in multiple pathological hallmarks of axon damage after experimental TBI.

Saphenous and sciatic nerve block to treat acute lower limb ischemic pain in the emergency department.

Acute limb ischemia (ALI) presents with significant pain that is often refractory to opioid pain management or is present in patients with relative contraindications to opioids. Here we present a case of ALI successfully managed with regional anesthesia using sciatic and saphenous nerve blocks. To our knowledge, this is the first case report of regional anesthesia for ALI performed in the Emergency Department (ED) by Emergency Medicine physicians.

SKOPE-Study of Ketorolac vs Opioid for Pain after Endoscopy: A Double-Blinded Randomized Control Trial in Patients Undergoing Ureteroscopy.

PURPOSE: Pain is the leading cause of unplanned emergency department visits and readmissions after ureteroscopy, making postoperative analgesic stewardship a priority given the current opioid epidemic. We conducted a double-blinded, randomized controlled trial, with noninferiority design, comparing nonsteroidal anti-inflammatory drugs to opiates for postoperative pain control in patients undergoing ureteroscopy for urolithiasis. MATERIALS AND METHODS: Patients were randomized and blinded to either oxycodone (5 mg) or ketorolac (10 mg), taken as needed, with 3 nonblinded oxycodone rescue pills for breakthrough pain. Primary study outcome was visual analogue scale pain score on postoperative days 1-5. Secondary outcomes included medication utilization, side effects, and Ureteral Stent Symptom Questionnaire scores. RESULTS: A total of 81 patients were included (43 oxycodone, 38 ketorolac). The 2 groups had comparable patient, stone, and perioperative characteristics. No differences were found in postoperative pain scores, study medication or rescue pill usage, or side effects. Higher maximum pain scores on days 1-5 (p <0.05) and higher questionnaire score (28.1 vs 21.7, p=0.045) correlated with analgesic usage, irrespective of treatment group. Patients receiving ketorolac reported significantly fewer days confined to bed (mean±SD 1.3±1.3 vs 2.3±2.6, p=0.02). There was no difference in unscheduled postoperative physician encounters. CONCLUSIONS: This is the first double-blinded randomized controlled trial comparing nonsteroidal anti-inflammatory drugs and opiates post-ureteroscopy, and demonstrates noninferiority of nonsteroidal anti-inflammatory drugs in pain control with similar efficacy, safety profile, physician contact and notably, earlier convalescence compared to the opioid group. This provides strong evidence against routine opioid use post-ureteroscopy, justifying continued investigation into reducing postoperative opiate prescriptions.

Drying Climates and Gendered Suffering: Links Between Drought, Food Insecurity, and Women's HIV in Less-Developed Countries.

HIV/AIDS represents the leading cause of death among women of reproductive age globally, and gender inequalities in the burden of HIV/AIDS are most pronounced in poorer countries. Drawing on ideas from feminist political ecology, we explore linkages between suffering from drought, food insecurity, and women's vulnerability to HIV. Using data from 91 less-developed countries, we construct a structural equation model to analyze the direct and indirect influence of these factors, alongside other socio-economic indicators, on the percentage of the adult population living with HIV that are women. We find that droughts are significant in shaping gender inequalities in the HIV burden indirectly through increased food insecurity. We draw on prior research to argue that due to gendered inequalities, food insecurity increases women's vulnerability to HIV by intensifying biological susceptibilities to the disease, reducing access to social and health resources, and motivating women to engage in risky sexual behaviors, such as transactional sex. Overall, our findings demonstrate that droughts serve as an important underlying factor in promoting HIV transmission among vulnerable women in poor countries, and that food insecurity is a key mechanism in driving this relationship.

Erythropoietin promotes functional recovery in a mouse model of postoperative ileus.

BACKGROUND: Dysmotility and postoperative ileus (POI) are major clinical problems after surgical trauma and it is associated with increased intestinal inflammation and oxidative stress. Despite the high occurrence of POI following intra-abdominal surgeries, no effective treatment is currently available. Erythropoietin (EPO) is a multifunctional tissue-protective cytokine with potent anti-inflammatory and anti-oxidative properties, and it is an FDA approved medicine for clinical use. While both EPO and EPO receptors (EPOR) are widely expressed in the gut, the role of EPO in POI is largely unknown. This study was designed to explore the possible beneficial effect of EPO in a mouse model of POI. METHODS: Mice were subjected to intestinal manipulation to induce standard POI and intestinal transit time was determined at 24-h post-injury with or without EPO treatment (5000 units/kg, once, IP, immediately after intestinal trauma). Intestinal samples were harvested for histological and immunohistochemical analysis. RESULTS: Systemic EPO significantly improved intestinal transit time compared with control group and it was associated with significantly increased levels of tissue macrophages and reduced levels of oxidative stress. CONCLUSIONS AND INFERENCES: This is the first pre-clinical study to document novel beneficial effects of EPO in gut dysmotility and our findings suggest that the beneficial effects of EPO in POI is predominantly mediated by its anti-oxidative and immunomodulatory properties.

The many roles of C1q.

The ability of a well-known component of the complement cascade to bind to a variety of receptors has implications for signaling biology, spinal cord injury and, possibly, the evolution of the complement system.

Human equivalent dose of oral 4-aminopyridine differentiates nerve crush injury from transection injury and improves post-injury function in mice.

4-Aminopyridine (4-AP), an FDA-approved drug for the symptomatic treatment of multiple sclerosis, is used to improve neuromuscular function in patients with diverse demyelinating disorders. We recently demonstrated that local, transdermal or injectable forms of 4-AP improve myelination, nerve conduction velocity, muscle atrophy, and motor function after traumatic peripheral nerve injury in mice. While oral 4-AP is most commonly used in the clinic, it is unknown whether human equivalent oral doses of 4-AP have effects on traumatic peripheral nerve injury differentiation, myelination, muscle atrophy, functional recovery, and post-injury inflammatory processes in animals. Mice with sciatic nerve crush or denervation injury received oral or intraperitoneal 4-AP (10 µg) or vehicle alone and were examined for pharmacokinetics, motor function, muscle mass, intrinsic muscle force, nerve morphological and gene expression profiles. 4-AP showed linear pharmacokinetics and the maximum plasma 4-AP concentrations were proportional to 4-AP dose. Acute single dose of oral 4-AP administration induced a rapid transient improvement in motor function that was different in traumatic peripheral nerve injury with or without nerve continuity, chronic daily oral 4-AP treatment significantly enhanced post crush injury motor function recovery and this effect was associated with improved myelination, muscle mass, and ex vivo muscle force. Polymerase chain reaction array analysis with crushed nerve revealed significant alterations in gene involved in axonal inflammation and regeneration. These findings provide convincing evidence that regardless of the route of administration, 4-AP can acutely differentiate traumatic peripheral nerve injury with or without nerve continuity and can enhance in vivo functional recovery with better preservation of myelin sheaths, muscle mass, and muscle force. The animal experiments were approved by the University Committee on Animal Research (UCAR) at the University of Rochester (UCAR-2009-019) on March 31, 2017.

Outcomes of Conservative Management of Splenic Injury Incurred During Percutaneous Nephrolithotomy.

Purpose: Splenic injury is a rare complication after left-sided percutaneous nephrolithotomy (PCNL). Although initial observation is often espoused, the natural history of nonoperative conservative management is not well established and the implications of splenic injury are not fully defined in this context. We sought to describe outcomes of conservative management of splenic injury incurred at PCNL. Patients and Methods: We performed a multi-institutional retrospective review of individual patients who underwent PCNL complicated by trans-splenic nephrostomy access injury. Demographic info, intraoperative data, management strategies, and outcomes were reviewed. Results: Nine patients suffered splenic injury after left PCNL. All patients had supracostal upper pole access under fluoroscopic guidance. Splenic injury was identified by computed tomography (CT) in the eight of nine (89%) who had imaging on first postoperative day. All eight patients were managed conservatively with nephrostomy dwell time of 2-21 days, one of whom (11%) required blood transfusion. The remaining patient (11%)-who had tubeless PCNL without postoperative imaging presented 5 days postoperatively with a delayed bleed and underwent emergent splenectomy. Seven of the nine (78%) were managed nonoperatively and without need for transfusion or embolization. Conclusion: The majority of patients incurring splenic injury during PCNL can be managed conservatively with maintenance of nephrostomy tube for ≥2 days. Consequences of unrecognized splenic injury may include splenic bleed and may prompt transfusion and/or splenectomy, underscoring role of routine postoperative CT to allow timely diagnosis, particularly in those undergoing upper pole supracostal left-sided percutaneous renal access.

Transdermal delivery of 4-aminopyridine accelerates motor functional recovery and improves nerve morphology following sciatic nerve crush injury in mice.

Oral 4-aminopyridine (4-AP) is clinically used for symptomatic relief in multiple sclerosis and we recently demonstrated that systemic 4-AP had previously unknown clinically-relevant effects after traumatic peripheral nerve injury including the promotion of re-myelination, improvement of nerve conductivity, and acceleration of functional recovery. We hypothesized that, instead of oral or injection administration, transdermal 4-AP (TD-4-AP) could also improve functional recovery after traumatic peripheral nerve injury. Mice with surgical traumatic peripheral nerve injury received TD-4AP or vehicle alone and were examined for skin permeability, pharmacokinetics, functional, electrophysiological, and nerve morphological properties. 4-AP showed linear pharmacokinetics and the maximum plasma 4-AP concentrations were proportional to TD-4-AP dose. While a single dose of TD-4-AP administration demonstrated rapid transient improvement in motor function, chronic TD-4-AP treatment significantly improved motor function and nerve conduction and these effects were associated with fewer degenerating axons and thicker myelin sheaths than those from vehicle controls. These findings provide direct evidence for the potential transdermal applicability of 4-AP and demonstrate that 4-AP delivered through the skin can enhance in-vivo functional recovery and nerve conduction while decreasing axonal degeneration. The animal experiments were approved by the University Committee on Animal Research (UCAR) at the University of Rochester (UCAR-2009-019) on March 31, 2017.

Ureteral Wall Injury with Ureteral Access Sheaths: A Randomized Prospective Trial.

Objective: To compare two commercially available ureteral access sheaths in their ability to access the renal collecting system and assess ureteral wall trauma using a prospective, randomized trial. Patients and Methods: Ninety-five patients undergoing ureteroscopy for renal stones were randomized to Cook Flexor™ or Boston Scientific Navigator HD™ 12/14F sheaths. If the initial sheath failed to advance, an alternate sheath was attempted. The primary outcome was the difference in these access sheaths to obtain access to the upper collecting system and the postoperative ureteral injury using standardized five-point classification system. Results: The overall success rate for sheath placement was 87.4% and did not differ for sheath groups. The Navigator HD was successful in 43% of the Flexor failures and was subjectively rated as easier to place (p = 0.018). Male gender, large stone burden, longer time of sheath insertion, and a more difficult subjective rating for sheath placement were associated with high-grade (grade 2 or 3) ureteral injury. Limitations include a small sample size and absence of long-term follow-up. Conclusion: Sheaths had equal success of placement and there was no significant difference in ureteral wall injury between the two sheaths. Subjectively difficult sheath placement and longer time of placement were associated with high-grade injury, suggesting that surgeons should carry a low threshold for switching to a smaller sheath when resistance is felt or if placement time is long. Clinical Trial number: Nct03349099.

Peripheral nerve injury and myelination: Potential therapeutic strategies.

Traumatic peripheral nerve injury represents a major clinical and public health problem that often leads to significant functional impairment and permanent disability. Despite modern diagnostic procedures and advanced microsurgical techniques, functional recovery after peripheral nerve repair is often unsatisfactory. Therefore, there is an unmet need for new therapeutic or adjunctive strategies to promote the functional recovery in nerve injury patients. In contrast to the central nervous system, Schwann cells in the peripheral nervous system play a pivotal role in several aspects of nerve repair such as degeneration, remyelination, and axonal growth. Several non-surgical approaches, including pharmacological, electrical, cell-based, and laser therapies, have been employed to promote myelination and enhance functional recovery after peripheral nerve injury. This review will succinctly discuss the potential therapeutic strategies in the context of myelination following peripheral neurotrauma.