The impact of hypertonic saline on damage control laparotomy after penetrating abdominal trauma.

PURPOSE: The inability to achieve primary fascial closure (PFC) after emergency laparotomy increases the rates of adverse outcomes including fistula formation, incisional hernia, and intraabdominal infection. Hypertonic saline (HTS) infusion improves early PFC rates and decreases time to PFC in patients undergoing damage control laparotomy (DCL) after injury. We hypothesized that in patients undergoing DCL after penetrating abdominal injury, HTS infusion would decrease the time to fascial closure as well as the volume of crystalloid required for resuscitation without inducing clinically relevant acute kidney injury (AKI) or electrolyte derangements. METHODS: We retrospectively analyzed all penetrating abdominal injury patients undergoing DCL within the University of Pennsylvania Health System (January 2015-December 2018). We compared patients who received 3% HTS at 30 mL/h (HTS) to those receiving isotonic fluid (ISO) for resuscitation while the abdominal fascia remained open. Primary outcomes were the rate of early PFC (PFC within 72 h) and time to PFC; secondary outcomes included acute kidney injury, sodium derangement, ventilator-free days, hospital length of stay (LOS), and ICU LOS. Intergroup comparisons occurred by ANOVA and Tukey's comparison, and student's t, and Fischer's exact tests, as appropriate. A Shapiro-Wilk test was performed to determine normality of distribution. RESULTS: Fifty-seven patients underwent DCL after penetrating abdominal injury (ISO n = 41, HTS n = 16). There were no significant intergroup differences in baseline characteristics or injury severity score. Mean time to fascial closure was significantly shorter in HTS (36.37 h ± 14.21 vs 59.05 h ± 50.75, p = 0.02), and the PFC rate was significantly higher in HTS (100% vs 73%, p = 0.01). Mean 24-h fluid and 48-h fluid totals were significantly less in HTS versus ISO (24 h: 5.2L ± 1.7 vs 8.6L ± 2.2, p = 0.01; 48 h: 1.3L ± 1.1 vs 2.6L ± 2.2, p = 0.008). During the first 72 h, peak sodium (Na) concentration (146.2 mEq/L ± 2.94 vs 142.8 mEq/L ± 3.67, p = 0.0017) as well as change in Na from ICU admission (5.1 mEq/L vs 2.3, p = 0.016) were significantly higher in HTS compared to ISO. Patients in the HTS group received significantly more blood in the trauma bay compared to ISO. There were no intergroup differences in intraoperative blood transfusion volume, AKI incidence, change in chloride concentration (△Cl) from ICU admit, Na to Cl gradient (Na:Cl), initial serum creatinine (Cr), peak post-operative Cr, change in creatinine concentration (△Cr) from ICU admission, creatinine clearance (CrCl), initial serum potassium (K), peak ICU K, change in K from ICU admission, initial pH, highest or lowest post-operative pH, mean hospital LOS, ICU LOS, and ventilator-free days. CONCLUSIONS: HTS infusion in patients undergoing DCL after penetrating abdominal injury decreases the time to fascial closure and led to 100% early PFC. HTS infusion also decreased resuscitative fluid volume without causing significant AKI or electrolyte derangement. HTS appears to offer a safe and effective fluid management approach in patients who sustain penetrating abdominal injury and DCL to support early PFC without inducing measurable harm. LEVEL OF EVIDENCE: Level III.

PlmCas12e (CasX2) cleavage of CCR5: impact of guide RNA spacer length and PAM sequence on cleavage activity.

Gene editing using CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) is under development as a therapeutic tool for the modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas9 systems from Streptococcus pyogenes and Staphylococcus aureus, alternative CRISPR systems have been identified from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of CRISPR/Cas enzymes. The Cas12e enzymes from non-pathogenic Deltaproteobacteria (CasX1, DpeCas12e) and Planctomycetes (CasX2, PlmCas12e) are smaller than Cas9, have a selective protospacer adjacent motif (PAM), and deliver a staggered cleavage cut with a 5-7 nucleotide overhang. We investigated the impact of guide RNA spacer length and alternative PAM sequences on cleavage activity to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes the CCR5 coreceptor used by human immunodeficiency virus-type 1 (HIV-1) to infect target cells. A 32 base-pair deletion in CCR5 (CCR5-[Formula: see text]32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR/Cas. We determined that CCR5 cleavage activity varied with the target site, spacer length, and the fourth nucleotide in the previously described PAM sequence, TTCN. Our analyses demonstrated a PAM preference for purines (adenine, guanine) over pyrimidines (thymidine, cytosine) in the fourth position of the CasX2 PAM. This improved understanding of CasX2 cleavage requirements facilitates the development of therapeutic strategies to recreate the CCR5-[Formula: see text]32 mutation in haematopoietic stem cells.

CAS12e (CASX2) CLEAVAGE OF CCR5: IMPACT OF GUIDE RNA LENGTH AND PAM SEQUENCE ON CLEAVAGE ACTIVITY.

CRISPR/Cas is under development as a therapeutic tool for the cleavage, excision, and/or modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9), alternative CRISPR systems have been identified using metagenomic datasets from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of gene editors. The Cas12e (CasX1, CasX2) endonucleases from non-pathogenic Deltaproteobacteria (DpeCas12e) and Planctomycetes (PlmCas12e) are more compact than SpCas9, have a more selective protospacer adjacent motif (PAM) requirement, and deliver a staggered cleavage cut with 5-7 base overhangs. We investigated varying guide RNA (spacer) lengths and alternative PAM sequences to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes one of two chemokine coreceptors required by HIV-1 to infect target cells, and a mutation of CCR5 (delta-32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR, TALENs, and ZFNs. We determined that CCR5 cleavage activity varied with the target site, guide RNA length, and the terminal nucleotide in the PAM sequence. Our analyses demonstrated a PlmCas12e PAM preference for purines (A, G) over pyrimidines (T, C) in the fourth position of the CasX2 PAM (TTCN). These analyses have contributed to a better understanding of CasX2 cleavage requirements and will position us more favorably to develop a therapeutic that creates the delta-32 mutation in the CCR5 gene in hematopoietic stem cells.

Long-Term Care Resident Awareness and Interest in Spasticity Treatments.

Spasticity is common in long-term care settings (affecting up to one in three residents), yet it remains under-treated despite safe and effective, Food and Drug Administration (FDA)-approved therapies. One barrier to treatment may be lack of awareness of available therapies for long-term care residents living with spasticity. A standardized spasticity treatment awareness and interest interview was conducted with 18 nursing home residents and 11 veterans' home residents in this cross-sectional study. Veterans' home residents were also asked about potential barriers to receiving spasticity treatment. Many residents across both long-term care facilities were unaware of most of the treatment options for spasticity. Participants were most aware of physical/occupational therapy (83%, 95% CI: 65-93%) and least aware of intrathecal baclofen (21%, 95% CI: 9-39%). After learning about treatments, only 7% of participants (95% CI: 0-23%) were not interested in receiving any form of spasticity treatment. Among residents previously unaware of spasticity treatments, at least one quarter became interested in receiving treatment and at least one-fifth indicated possibly being interested in the treatment after learning about it. Potential barriers to receiving treatment included traveling to see a doctor and limited knowledge of insurance coverage of spasticity treatments. These results suggest that patient-centered approaches, including education and discerning patient preferences, may improve spasticity treatment in long-term care settings.

Analysis of CRISPR/Cas9 Guide RNA Efficiency and Specificity Against Genetically Diverse HIV-1 Isolates.

Gene editing approaches using CRISPR/Cas9 are being developed as a means for targeting the integrated HIV-1 provirus. Enthusiasm for the use of gene editing as an anti-HIV-1 therapeutic has been tempered by concerns about the specificity and efficacy of this approach. Guide RNAs (gRNAs) that target conserved sequences across a wide range of genetically diverse HIV-1 isolates will have greater clinical utility. However, on-target efficacy should be considered in the context of off-target cleavage events as these may comprise an essential safety parameter for CRISPR-based therapeutics. We analyzed a panel of Streptococcus pyogenes Cas9 (SpCas9) gRNAs directed to the 5' and 3' long terminal repeat (LTR) regions of HIV-1. We used in vitro cleavage assays with genetically diverse HIV-1 LTR sequences to determine gRNA activity across HIV-1 clades. Lipid-based transfection of gRNA/Cas9 ribonucleoproteins was used to assess targeting of the integrated HIV-1 proviral sequence in cells (in vivo). For both the in vitro and in vivo experiments, we observed increased efficiency of sequence disruption through the simultaneous use of two distinct gRNAs. Next, CIRCLE-Seq was utilized to identify off-target cleavage events using genomic DNA from cells with integrated HIV-1 proviral DNA. We identified a gRNA targeting the U3 region of the LTR (termed SpCas9-127HBX2) with broad cleavage efficiency against sequences from genetically diverse HIV-1 strains. Based on these results, we propose a workflow for identification and development of anti-HIV CRISPR therapeutics.

Effectiveness of perioperative antiepileptic drug prophylaxis for early and late seizures following oncologic neurosurgery: a meta-analysis.

OBJECTIVE: The purpose of this meta-analysis was to evaluate the impact of perioperative antiepileptic drug (AED) prophylaxis on short- and long-term seizure incidence among patients undergoing brain tumor surgery. It is the first meta-analysis to focus exclusively on perioperative AED prophylaxis among patients undergoing brain tumor surgery. METHODS: The authors searched PubMed/MEDLINE, Embase, Cochrane Central Register of Controlled Trials, clinicaltrials.gov, and the System for Information on Gray Literature in Europe for records related to perioperative AED prophylaxis for patients with brain tumors. Risk of bias in the included studies was assessed using the Cochrane risk of bias tool. Incidence rates for early seizures (within the first postoperative week) and total seizures were estimated based on data from randomized controlled trials. A Mantel-Haenszel random-effects model was used to analyze pooled relative risk (RR) of early seizures (within the first postoperative week) and total seizures associated with perioperative AED prophylaxis versus control. RESULTS: Four RCTs involving 352 patients met the criteria of inclusion. The results demonstrated that perioperative AED prophylaxis for patients undergoing brain tumor surgery provides a statistically significant reduction in risk of early postoperative seizures compared with control (RR = 0.352, 95% confidence interval 0.130-0.949, p = 0.039). AED prophylaxis had no statistically significant effect on the total (combined short- and long-term) incidence of seizures. CONCLUSIONS: This meta-analysis demonstrates for the first time that perioperative AED prophylaxis for brain tumor surgery provides a statistically significant reduction in early postoperative seizure risk.

Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175.

A member of a Plasmodium receptor family for erythrocyte invasion was identified on chromosome 13 from the Plasmodium falciparum genome sequence of the Sanger Centre (Cambridge, U.K.). The protein (named BAEBL) has homology to EBA-175, a P. falciparum receptor that binds specifically to sialic acid and the peptide backbone of glycophorin A on erythrocytes. Both EBA-175 and BAEBL localize to the micronemes, organelles at the invasive ends of the parasites that contain other members of the family. Like EBA-175, the erythrocyte receptor for BAEBL is destroyed by neuraminidase and trypsin, indicating that the erythrocyte receptor is a sialoglycoprotein. Its specificity, however, differs from that of EBA-175 in that BAEBL can bind to erythrocytes that lack glycophorin A, the receptor for EBA-175. It has reduced binding to erythrocytes with the Gerbich mutation found in another erythrocyte, sialoglycoprotein (glycophorin C/D). The interest in BAEBL's reduced binding to Gerbich erythrocytes derives from the high frequency of the Gerbich phenotype in some regions of Papua New Guinea where P. falciparum is hyperendemic.

Identification of a Plasmodium falciparum intercellular adhesion molecule-1 binding domain: a parasite adhesion trait implicated in cerebral malaria.

Binding of infected erythrocytes to brain venules is a central pathogenic event in the lethal malaria disease complication, cerebral malaria. The only parasite adhesion trait linked to cerebral sequestration is binding to intercellular adhesion molecule-1 (ICAM-1). In this report, we show that Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) binds ICAM-1. We have cloned and expressed PfEMP1 recombinant proteins from the A4tres parasite. Using heterologous expression in mammalian cells, the minimal ICAM-1 binding domain was a complex domain consisting of the second Duffy binding-like (DBL) domain and the C2 domain. Constructs that contained either domain alone did not bind ICAM-1. Based on phylogenetic criteria, there are five distinct PfEMP1 DBL types designated alpha, beta, gamma, delta, and epsilon. The DBL domain from the A4tres that binds ICAM-1 is DBLbeta type. A PfEMP1 cloned from a distinct ICAM-1 binding variant, the A4 parasite, contains a DBLbeta domain and a C2 domain in tandem arrangement similar to the A4tres PfEMP1. Anti-PfEMP1 antisera implicate the DBLbeta domain from A4var PfEMP1 in ICAM-1 adhesion. The identification of a P. falciparum ICAM-1 binding domain may clarify mechanisms responsible for the pathogenesis of cerebral malaria and lead to interventions or vaccines that reduce malarial disease.

Analysis of adhesive domains from the A4VAR Plasmodium falciparum erythrocyte membrane protein-1 identifies a CD36 binding domain.

The A4VAR is a variant antigen expressed by a clonal line that binds CD36 and intercellular adhesion molecule-1, ICAM-1. We have cloned and sequenced the extracellular domain coded by the A4var gene. To probe the relationship between A4var expression and parasite adhesion to ICAM-1, var mRNA and protein expression were analyzed in an enriched population of A4 parasites that displayed higher ICAM-1 binding. By Northern analyses, A4var was the predominant var message and antisera raised against a recombinant A4VAR protein reacted with the majority of infected erythrocytes, reinforcing previous conclusions that A4VAR binds ICAM-1. A4VAR contains five Duffy-binding like (DBL) domains, and two cysteine-rich interdomain regions (CIDR) domains. DBL and CIDR domains from A4VAR were expressed in mammalian cells to determine which regions mediate binding to CD36 and ICAM-1. Using several different binding assays, the A4VAR CIDR1 was the only domain found to bind CD36. In contrast, the same assays were unable to identify the ICAM-1 binding domain in A4VAR. This is the first time that each of the DBL and CIDR domains from a Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) have been systematically expressed and tested for binding. These results confirm that CIDR1 is sufficient to bind CD36 without any apparent contribution from other domains.

Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes.

Plasmodium falciparum expresses on the host erythrocyte surface clonally variant antigens and ligands that mediate adherence to endothelial receptors. Both are central to pathogenesis, since they allow chronicity of infection and lead to concentration of infected erythrocytes in cerebral vessels. Here we show that expression of variant antigenic determinants is correlated with expression of individual members of a large, multigene family named var. Each var gene contains copies of a motif that has been previously shown to bind diverse host receptors; expression of a specific var gene correlated with binding to ICAM-1. Thus, our findings are consistent with the involvement of var genes in antigenic variation and binding to endothelium.

Plasmodium falciparum protein associated with the invasion junction contains a conserved oxidoreductase domain.

The merozoite cap protein-1 (MCP-1) of Plasmodium falciparum follows the distribution of the moving junction during invasion of erythrocytes. We have cloned the gene encoding this protein from a cDNA library using a monoclonal antibody. The protein lacks a signal sequence and has no predicted transmembrane domains; none of the antisera reacts with the surfaces of intact merozoites, indicating that the cap distribution is submembranous. MCP-1 is divided into three domains. The N-terminal domain includes a 52-amino-acid region that is highly conserved in a large family of bacterial and eukaryotic proteins. Based on the known functions of two proteins of this family and the pattern of amino acid conservation, it is predicted that this domain may possess oxido-reductase activity, since the active cysteine residue of this domain is invariant in all proteins of the family. The other two domains of MCP-1 are not found in any other members of this protein family and may reflect the specific function of MCP-1 in invasion. The middle domain is negatively charged and enriched in glutamate; the C-terminal domain is positively charged and enriched in lysine. By virtue of its positive charge, the C-terminal domain resembles domains in some cytoskeleton-associated proteins and may mediate the interaction of MCP-1 with cytoskeleton in Plasmodium.