Whole-brain annotation and multi-connectome cell typing of Drosophila.

The fruit fly Drosophila melanogaster has emerged as a key model organism in neuroscience, in large part due to the concentration of collaboratively generated molecular, genetic and digital resources available for it. Here we complement the approximately 140,000 neuron FlyWire whole-brain connectome1 with a systematic and hierarchical annotation of neuronal classes, cell types and developmental units (hemilineages). Of 8,453 annotated cell types, 3,643 were previously proposed in the partial hemibrain connectome2, and 4,581 are new types, mostly from brain regions outside the hemibrain subvolume. Although nearly all hemibrain neurons could be matched morphologically in FlyWire, about one-third of cell types proposed for the hemibrain could not be reliably reidentified. We therefore propose a new definition of cell type as groups of cells that are each quantitatively more similar to cells in a different brain than to any other cell in the same brain, and we validate this definition through joint analysis of FlyWire and hemibrain connectomes. Further analysis defined simple heuristics for the reliability of connections between brains, revealed broad stereotypy and occasional variability in neuron count and connectivity, and provided evidence for functional homeostasis in the mushroom body through adjustments of the absolute amount of excitatory input while maintaining the excitation/inhibition ratio. Our work defines a consensus cell type atlas for the fly brain and provides both an intellectual framework and open-source toolchain for brain-scale comparative connectomics.

Extended Lymph Node Sampling During Surgery for Pediatric Renal Tumors Concerning for Malignancy Does Not Increase Postoperative Complication Rates.

PURPOSE: Although Children's Oncology Group renal tumor protocols mandate lymph node sampling during extirpative surgery for pediatric renal tumors, lymph node sampling is often omitted or low yield. Concerns over morbidity associated with extended lymph node sampling have led to hesitancy in adopting a formal lymph node sampling template. We hypothesized that complications in children undergoing lymph node sampling for renal tumors would be rare, and not associated with the number of lymph nodes sampled. MATERIALS AND METHODS: A single-institution, retrospective review of patients aged 0-18 years undergoing extirpative renal surgery with lymph node sampling for a suspected malignancy between 2005 and 2019 was performed. Patients with 0 or an unknown number of lymph nodes sampled or <150 days of follow-up were excluded. A "clinically significant" complication was defined as any Clavien complication ≥III, small-bowel obstruction, chylous ascites, organ injury, or wound infection. The number of lymph nodes sampled and its influence on the odds of experiencing a clinically significant complication was examined. RESULTS: A total of 144 patients met inclusion criteria. Median patient age was 38 months. Twenty-one patients (15%) had a clinically significant complication, the most common of which was ileus/small-bowel obstruction (n=16). In a multivariable analysis, increased lymph node yield was not found to influence the odds of experiencing a clinically significant complication (P = .6). CONCLUSIONS: In this cohort, there was no statistically significant difference in clinically significant complications in patients who underwent more extensive lymph node sampling during surgery for a suspected malignant pediatric renal tumor. Future studies on protocol adherence, staging accuracy, and survival trends using a lymph node sampling template in these patients should be performed.

Pediatric Small Renal Masses: Can Tumor Size Predict Histology and the Potential for Nephron-sparing Surgery?

PURPOSE: The majority of children with unilateral renal masses suspicious for malignancy undergo radical nephrectomy, while nephron-sparing surgery is reserved for select cases. We investigated the impact of tumor size on the probability of histology. We hypothesized that pediatric small renal masses are more likely benign or non-Wilms tumor, thus potentially appropriate for nephron-sparing surgery. MATERIALS AND METHODS: The SEER (Surveillance, Epidemiology, and End Results) database was analyzed for patients aged 0-18 years diagnosed with a unilateral renal mass from 2000-2016. Statistical analysis was performed to help determine a tumor size cut point to predict Wilms tumor and assess the predictive value of tumor size on Wilms tumor histology. Additionally, a retrospective review was performed of patients 0-18 years old who underwent surgery for a unilateral renal mass at a single institution from 2005-2019. Statistical analysis was performed to assess the predictive value of tumor size on final histology. RESULTS: From the SEER analysis, 2,016 patients were included. A total of 1,672 tumors (82.9%) were Wilms tumor. Analysis revealed 4 cm to be a suitable cut point to distinguish non-Wilms tumor. Tumors ≥4 cm were more likely Wilms tumor (OR 2.67, P ≤ .001), but this was driven by the statistical significance in children 5-9 years old. From the institutional analysis, 134 patients were included. Ninety-seven tumors (72.3%) were Wilms tumor. Tumors ≥4 cm had higher odds of being Wilms tumor (OR 30.85, P = .001), malignant (OR 6.75, P = .005), and having radical nephrectomy-appropriate histology (OR 46.79, P < .001). CONCLUSIONS: The probability that a pediatric unilateral renal mass is Wilms tumor increases with tumor size. Four centimeters is a logical cut point to start the conversation around defining pediatric small renal masses and may help predict nephron-sparing surgery-appropriate histology.

Enhanced recovery after surgery and anesthetic outcomes in pediatric reconstructive urologic surgery.

PURPOSE: Enhanced recovery after surgery (ERAS) is a perioperative management strategy to hasten postoperative recovery. We examined the effects of a pilot implementation of ERAS for pediatric patients on anesthetic outcomes. METHODS: We performed a prospective case-control study utilizing an ERAS protocol in patients aged < 18 years undergoing urologic reconstruction that included a bowel anastomosis. Protocol elements included: multimodal analgesia, opioid minimization, and routine nausea/vomiting prophylaxis. ERAS patients were propensity-matched with historical controls. Outcomes of interest included maximum PACU pain score, time to first opioid, opioid-free days, and need for opioids on day of discharge. RESULTS: A total of 13 ERAS patients and 26 historical controls were included, with median ages 9.9 years (IQR 9.1-11) and 10.4 years (IQR 8.0-12.4), respectively. ERAS increased the percentage of patients who did not receive any intraoperative or postoperative opioids (0% vs 15%, p = 0.046 for both) and reduced maximum PACU pain score (3 vs 0, p < 0.001). The use of postoperative supplemental oxygen was decreased in the ERAS group (85% vs 38%, p = 0.013). CONCLUSIONS: The implementation of an ERAS protocol appears to decrease postoperative pain, opioid usage, and positively impact other anesthetic outcomes in children undergoing urologic reconstructive surgery utilizing a bowel anastomosis.

Mesenchymal Stem Cells Delivered Locally to Ischemia-Reperfused Kidneys via Injectable Hyaluronic Acid Hydrogels Decrease Extracellular Matrix Remodeling 1 Month after Injury in Male Mice.

The translation of stem cell therapies has been hindered by low cell survival and retention rates. Injectable hydrogels enable the site-specific delivery of therapeutic cargo, including cells, to overcome these challenges. We hypothesized that delivery of mesenchymal stem cells (MSC) via shear-thinning and injectable hyaluronic acid (HA) hydrogels would mitigate renal damage following ischemia-reperfusion acute kidney injury. Acute kidney injury (AKI) was induced in mice by bilateral or unilateral ischemia-reperfusion kidney injury. Three days later, mice were treated with MSCs either suspended in media injected intravenously via the tail vein, or injected under the capsule of the left kidney, or MSCs suspended in HA injected under the capsule of the left kidney. Serial measurements of serum and urine biomarkers of renal function and injury, as well as transcutaneous glomerular filtration rate (tGFR) were performed. In vivo optical imaging showed that MSCs localized to both kidneys in a sustained manner after bilateral ischemia and remained within the ipsilateral treated kidney after unilateral ischemic AKI. One month after injury, MSC/HA treatment significantly reduced urinary NGAL compared to controls; it did not significantly reduce markers of fibrosis compared to untreated controls. An analysis of kidney proteomes revealed decreased extracellular matrix remodeling and high overlap with sham proteomes in MSC/HA-treated animals. Hydrogel-assisted MSC delivery shows promise as a therapeutic treatment following acute kidney injury.

Whole-brain annotation and multi-connectome cell typing quantifies circuit stereotypy in Drosophila.

The fruit fly Drosophila melanogaster combines surprisingly sophisticated behaviour with a highly tractable nervous system. A large part of the fly's success as a model organism in modern neuroscience stems from the concentration of collaboratively generated molecular genetic and digital resources. As presented in our FlyWire companion paper 1 , this now includes the first full brain connectome of an adult animal. Here we report the systematic and hierarchical annotation of this ~130,000-neuron connectome including neuronal classes, cell types and developmental units (hemilineages). This enables any researcher to navigate this huge dataset and find systems and neurons of interest, linked to the literature through the Virtual Fly Brain database 2 . Crucially, this resource includes 4,552 cell types. 3,094 are rigorous consensus validations of cell types previously proposed in the hemibrain connectome 3 . In addition, we propose 1,458 new cell types, arising mostly from the fact that the FlyWire connectome spans the whole brain, whereas the hemibrain derives from a subvolume. Comparison of FlyWire and the hemibrain showed that cell type counts and strong connections were largely stable, but connection weights were surprisingly variable within and across animals. Further analysis defined simple heuristics for connectome interpretation: connections stronger than 10 unitary synapses or providing >1% of the input to a target cell are highly conserved. Some cell types showed increased variability across connectomes: the most common cell type in the mushroom body, required for learning and memory, is almost twice as numerous in FlyWire as the hemibrain. We find evidence for functional homeostasis through adjustments of the absolute amount of excitatory input while maintaining the excitation-inhibition ratio. Finally, and surprisingly, about one third of the cell types proposed in the hemibrain connectome could not yet be reliably identified in the FlyWire connectome. We therefore suggest that cell types should be defined to be robust to inter-individual variation, namely as groups of cells that are quantitatively more similar to cells in a different brain than to any other cell in the same brain. Joint analysis of the FlyWire and hemibrain connectomes demonstrates the viability and utility of this new definition. Our work defines a consensus cell type atlas for the fly brain and provides both an intellectual framework and open source toolchain for brain-scale comparative connectomics.

Ureteral calculi detection using low dose computerized tomography protocols is compromised in overweight and underweight patients.

PURPOSE: Low dose computerized tomography protocols have demonstrated a reduction in radiation exposure while maintaining excellent sensitivity and specificity in the detection of stones in patients of average size. Low dose computerized tomography protocols have not yet been evaluated in subjects in the extremes of weight. We evaluated the effect of body weight when using low dose protocols to detect ureteral calculi. MATERIALS AND METHODS: Three cadavers of increasing weight (55, 85 and 115 kg) were prepared by inserting 721 calcium oxalate stones (range 3 to 7 mm) in 33 random configurations into urinary tracts. Cadavers were then scanned using a GE LightSpeed® at 7 radiation settings. An independent, blinded review by a radiologist was conducted to generate ROC curves, with areas under the curve compared using a 1-way ANOVA (α = 0.05). RESULTS: Sensitivity and specificity were significantly lower in the low and high weight cadavers compared to the medium weight cadaver at 5 mAs (p <0.001) and 7.5 mAs (p = 0.048). Differences in sensitivity and specificity at radiation settings of 15 mAs or greater were not significant. CONCLUSIONS: The sensitivity and specificity for the detection of ureteral calculi on computerized tomography were decreased for underweight and overweight subjects when using extremely low dose radiation settings (less than 1 mSv). Low dose protocols of 15 mAs (2 mSv) can still be used for these subjects without jeopardizing the ability to identify ureteral stones.

Outcomes of dialysis in neonates with anuric end-stage renal disease at birth: ethical considerations.

INTRODUCTION: We present a case series of neonates with anuric ESRD undergoing renal replacement therapy (RRT) and discuss the associated ethical implications of RRT in this population. METHODS: We reviewed patients who initiated RRT within 1 week of life due to anuric ESRD from 2009-2019 at a single tertiary center. Primary outcomes were receipt of renal transplant (RT), one-year survival, and overall survival. RESULTS: Five patients met the inclusion criteria. Two patients received an RT. One-year survival was 80%, while overall survival was 60% with a median follow-up of 18 months. In the 2 still-living patients who have not undergone RT, they are ineligible, one due to recent malignancy and the other from acquired cardiovascular comorbidities. CONCLUSION: Patients with anuric ESRD requiring RRT undergo multiple treatment challenges with low RT and survival rates. These findings should be shared with families considering intervention for cases of severe renal disease diagnosed prenatally.

A dynamic model of membrane-bound phospholipase Cß2 activation by Gßγ subunits.

Phospholipase C (PLC) ß2, a well studied member of the family of enzymes that catalyze the hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into secondary messengers, can be activated by the Gßγ subunits of heterotrimeric G-proteins in a manner that depends on the presence and composition of the associated phospholipid membrane surface. The N-terminal pleckstrin homology (PH) domain of PLCß2 mediates both the response to Gßγ and membrane binding, but how these interactions are coupled to yield an activated catalytic core remains unknown. Here we propose a mechanism based on molecular models of truncated PLCß2 in its activated form complexed with Gßγ and in the catalytically inactive/membrane-bound form, obtained with the application of protein-protein docking algorithms and coarse-grained molecular dynamics simulations. These models were probed experimentally, and the inferences were confirmed by results from a combination of molecular biology and fluorescence assays. Results from the dynamic simulations of the molecular models and their interactions with various lipid bilayers identify the determinants of PLCß2-PH domain specificity for Gßγ and lipid membranes and suggest a mechanism for the previously reported dependence of Gßγ activation on the associated membrane composition. Together, these findings explain the roles of the different activators in terms of their effect on the orientations of the PH and catalytic core domains relative to the lipid membranes.

A Cystic Renal Mass in the Setting of a Pneumothorax: More Than Meets the Eye?

DICER1 syndrome is a rare hereditary cancer predisposition syndrome that has relevance to pediatric urology providers due to its association with many various pediatric genitourinary malignancies. We describe the case of a pediatric patient who was eventually diagnosed with a pathogenic DICER1 germline variant after undergoing resection of a cystic nephroma and pleuropulmonary blastoma.

Membrane localization is critical for activation of the PICK1 BAR domain.

The PSD-95/Discs-large/ZO-1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C-terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N-terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain-dependent redistribution to clusters colocalizing with markers of recycling endosomal compartments. A similar clustering was observed both upon truncation of a short putative alpha-helical segment in the linker between the PDZ and the BAR domains and upon coexpression of PICK1 with a transmembrane PDZ ligand, including the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit, the GluR2 C-terminus transferred to the single transmembrane protein Tac or the dopamine transporter C-terminus transferred to Tac. In contrast, transfer of the GluR2 C-terminus to cyan fluorescent protein, a cytosolic protein, did not elicit BAR domain-dependent clustering. Instead, localizing PICK1 to the membrane by introducing an N-terminal myristoylation site produced BAR domain-dependent, but ligand-independent, PICK1 clustering. The data support that in the absence of PDZ ligand, the PICK1 BAR domain is inhibited through a PDZ domain-dependent and linker-dependent mechanism. Moreover, they suggest that unmasking of the BAR domain's membrane-binding capacity is not a consequence of ligand binding to the PDZ domain per se but results from, and coincides with, recruitment of PICK1 to a membrane compartment.

Reflux Timing Is a Predictor of Successful Endoscopic Treatment of Vesicoureteral Reflux.

OBJECTIVE: To determine whether vesicoureteral reflux (VUR) that occurs during either the filling or voiding phase on voiding cystourethrogram (VCUG) has prognostic significance on successful endoscopic treatment. MATERIALS AND METHODS: A retrospective review was performed of 299 patients (438 ureters) with VUR who underwent endoscopic treatment with dextranomer/hyaluronic acid copolymer (Deflux) at a single institution from 2010 to 2013. Success was defined as absence of VUR on 3-month follow-up VCUG. Preoperative VCUGs were analyzed to determine whether the onset of VUR occurred during the filling or voiding phase. Predictor variables to determine success were analyzed, with a specific focus on VUR timing. RESULTS: Success rate was 319/438 (72.8%) by ureter and 202/299 (67.6%) by patient. Reflux was seen during the filling and voiding phases in 290 and 148 ureters, respectively. Success rate was 203/290 (78%) for filling VUR and 116/148 (70%) for voiding VUR. Univariable analysis revealed voiding VUR had significantly increased odds of success (odds ratio [OR] 3.2, P = .049), while high-grade reflux (OR 0.53, P = .005) had significantly decreased odds of success. Multivariable analysis showed that voiding VUR (OR 3.2, P = .005) had significantly higher odds of success while those with high grade reflux (OR 0.42, P = .017) had significantly decreased odds of success. CONCLUSION: The timing of VUR on preoperative VCUG appears to be an important independent predictor of successful endoscopic treatment of VUR. This has important clinical considerations when selecting VUR patients who would be best candidates for endoscopic treatment.

Active state-like conformational elements in the beta2-AR and a photoactivated intermediate of rhodopsin identified by dynamic properties of GPCRs.

G-Protein-coupled receptors (GPCRs) adopt various functionally relevant conformational states in cell signaling processes. Recently determined crystal structures of rhodopsin and the beta 2-adrenergic receptor (beta 2-AR) offer insight into previously uncharacterized active conformations, but the molecular states of these GPCRs are likely to contain both inactive and active-like conformational elements. We have identified conformational rearrangements in the dynamics of the TM7-HX8 segment that relate to the properties of the conserved NPxxY(x)5,6F motif and show that they can be used to identify active state-like conformational elements in the corresponding regions of the new structures of rhodopsin and the beta 2-AR.

Outpatient Percutaneous Nephrolithotomy: The UC San Diego Health Experience.

INTRODUCTION: Outpatient percutaneous nephrolithotomy (PCNL) has been described for highly selected patients. We sought to assess the safety and feasibility of outpatient PCNL in a tertiary referral stone center without strict patient selection criteria. MATERIALS AND METHODS: We reviewed all PCNLs performed at our institution from September 2015 to October 2016. Of the 97 eligible cases, 60 patients underwent planned outpatient PCNL. Primary outcome was complication rate, and secondary outcome determined predictor variables of inpatient admission. RESULTS: Thirty-seven inpatient and 60 planned outpatient (one bilateral) PCNLs were performed with 65% and 44% American Society of Anesthesiologists (ASA) score ≥3, respectively. The 30-day overall complication rate for the inpatient and planned outpatient groups was 27% and 20%, respectively (p = 0.43) [70% and 92% Clavien grades I-II]. Emergency department presentation within 30 days was 19% and 18% (p = 0.94), and unplanned hospital readmission rate was 3% and 10% (p = 0.05). The 37 inpatient PCNL patients had larger total stone burden than outpatient cases (40.7 vs 25.8 mm, p = 0.0014); more often required two or more punctures into the kidney during the procedure (73% vs 45%, p = 0.025); and more often had supracostal access (20% vs 7%, p = 0.05). For the outpatient PCNL cohort, 72% patients were discharged same day, 28% were observed overnight for refractory symptoms or social reasons. Outpatient cohort radiographic stone-free rate by CT (no stones) was 67%. CONCLUSION: Outpatient PCNL has been safely and effectively performed within our institution in moderate-sized stones almost regardless of comorbidity status. We suggest that this approach is a potential algorithmic change in centers with sufficient case volume.

Synthesis, characterization, and in vivo efficacy of shell cross-linked nanoparticle formulations carrying silver antimicrobials as aerosolized therapeutics.

The use of nebulizable, nanoparticle-based antimicrobial delivery systems can improve efficacy and reduce toxicity for treatment of multi-drug-resistant bacteria in the chronically infected lungs of cystic fibrosis patients. Nanoparticle vehicles are particularly useful for applying broad-spectrum silver-based antimicrobials, for instance, to improve the residence time of small-molecule silver carbene complexes (SCCs) within the lung. Therefore, we have synthesized multifunctional, shell cross-linked knedel-like polymeric nanoparticles (SCK NPs) and capitalized on the ability to independently load the shell and core with silver-based antimicrobial agents. We formulated three silver-loaded variants of SCK NPs: shell-loaded with silver cations, core-loaded with SCC10, and combined loading of shell silver cations and core SCC10. All three formulations provided a sustained delivery of silver over the course of at least 2-4 days. The two SCK NP formulations with SCC10 loaded in the core each exhibited excellent antimicrobial activity and efficacy in vivo in a mouse model of Pseudomonas aeruginosa pneumonia. SCK NPs with shell silver cation-load only, while efficacious in vitro, failed to demonstrate efficacy in vivo. However, a single dose of core SCC10-loaded SCK NPs (0.74 ± 0.16 mg Ag) provided a 28% survival advantage over sham treatment, and administration of two doses (0.88 mg Ag) improved survival to 60%. In contrast, a total of 14.5 mg of Ag(+) delivered over 5 doses at 12 h intervals was necessary to achieve a 60% survival advantage with a free-drug (SCC1) formulation. Thus, SCK NPs show promise for clinical impact by greatly reducing antimicrobial dosage and dosing frequency, which could minimize toxicity and improve patient adherence.

Hybrid transureteral natural orifice translumenal endoscopic nephrectomy: a feasibility study in the porcine model.

BACKGROUND AND PURPOSE: Natural orifice approaches for nephrectomy have included access via the stomach, vagina, bladder, and rectum. The use of the ureter as a natural orifice for natural orifice translumenal endoscopic surgery (NOTES) nephrectomy has not been previously reported. The purpose of this study is to test the feasibility of transureteral laparoscopic NOTES nephrectomy. MATERIALS AND METHODS: Three female farm pigs (29.2-30.8 kg) were placed into the lithotomy position. A cystoscopically placed extra-stiff guidewire was used to place a prototype dilating sheath into the left ureter. After dilation of the ureter and urethra, the sheath was exchanged for a 12-mm bariatric laparoscopic trocar. A 10.5-inch long 10-mm offset operating laparoscope with an internal 5-mm working port was used for the nephrectomy. One 2-mm and one 2/3-mm port were placed transabdominally to facilitate in situ morcellation. The kidney was cut into slices using the bipolar device and extracted via the ureteral port using the housing of a 12-mm bariatric stapling device. RESULTS: All three transureteral nephrectomies were successfully completed. The total mean operative time was 220 minutes (range 113-346 min). Component portions of the procedure were: Ureteral access (mean 21 min), nephrectomy (mean 70 min), and kidney morcellation (mean 103 min). Mean estimated blood loss was 20 mL (range 5-50 mL). There were no intraoperative complications. CONCLUSIONS: This nonsurvival porcine feasibility study demonstrates the successful performance of transureteral nephrectomy. This approach shows promise as a way to decrease the invasiveness of NOTES nephrectomy by using the ureteral orifice as an access site.

A theobromine derived silver N-heterocyclic carbene: synthesis, characterization, and antimicrobial efficacy studies on cystic fibrosis relevant pathogens.

The increasing incidence of multidrug-resistant (MDR) pulmonary infections in the cystic fibrosis (CF) population has prompted the investigation of innovative silver based therapeutics. The functionalization of the naturally occurring xanthine theobromine at the N(1) nitrogen atom with an ethanol substituent followed by the methylation of the N(9) nitrogen atom gives the N-heterocyclic carbene precursor 1-(2-hydroxyethyl)-3,7,9-trimethylxanthinium iodide. The reaction of this xanthinium salt with silver acetate produces the highly hydrophilic silver carbene complex SCC8. The in vitro antimicrobial efficacy of this newly synthesized complex was evaluated with excellent results on a variety of virulent and MDR pathogens isolated from CF patients. A comparative in vivo study between the known caffeine derived silver carbene SCC1 and SCC8 demonstrated the ability of both complexes to improve the survival rates of mice in a pneumonia model utilizing the clinically isolated infectious strain of Pseudomonas aeruginosa PA M57-15.

The antimicrobial efficacy of sustained release silver-carbene complex-loaded L-tyrosine polyphosphate nanoparticles: characterization, in vitro and in vivo studies.

The pressing need to treat multi-drug resistant bacteria in the chronically infected lungs of cystic fibrosis (CF) patients has given rise to novel nebulized antimicrobials. We have synthesized a silver-carbene complex (SCC10) active against a variety of bacterial strains associated with CF and chronic lung infections. Our studies have demonstrated that SCC10-loaded into L-tyrosine polyphosphate nanoparticles (LTP NPs) exhibits excellent antimicrobial activity in vitro and in vivo against the CF relevant bacteria Pseudomonas aeruginosa. Encapsulation of SCC10 in LTP NPs provides sustained release of the antimicrobial over the course of several days translating into efficacious results in vivo with only two administered doses over a 72 h period.