Trauma and pregnancy: Is flow cytometry detection and quantification of fetal red blood cells useful?

OBJECTIVE: To assess whether positive flow cytometry quantification of fetal red blood cells is associated with adverse maternal and neonatal outcomes in cases of mild trauma during pregnancy. STUDY DESIGN: A retrospective database study was conducted at a single tertiary center between 2013 and 2019. All pregnant women with viable gestation involved in trauma who underwent flow cytometry quantification of fetal red blood cells were included in the study. Flow cytometry was considered positive (≥0.03/≥30 ml). Composite adverse maternal and neonatal outcome was defined as one or more of the following: intrauterine fetal death, placental abruption, pre-term birth <37 weeks of gestation, immediate premature rupture of the membranes, and immediate delivery following trauma. Univariate analysis was performed followed by multivariate logistic regression analysis controlling for potential confounders, to assess the role of flow cytometry in predicting adverse maternal and neonatal outcome. Adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were calculated. RESULTS: During the study period 1023 women met inclusion and exclusion criteria. The mechanisms of injury were motor vehicle accident in 387 women (38%), falls in 367 (36%), direct abdominal injury in 353 (35%) and in 14 women (1%) other mechanism of injury. Flow cytometry was considered positive (≥0.03/≥30 ml) in 119 women (11.6%) with median result of 0.03 [0.03-0.04], and negative in 904 women (88.4%) ((≤0.03/≤30 ml) with median result of 0.01 [0.01-0.02]. Composite adverse outcome occurred in 8% of the women involved in trauma during pregnancy, with no difference between the groups with vs. without positive flow cytometry (4.2% vs. 8.5%; p = 0.1). Positive flow cytometry was not associated with any adverse maternal or neonatal outcome. This was confirmed on multivariate analysis controlling for potential confounders. CONCLUSION: Flow cytometry result is not related to adverse maternal and fetal/neonatal outcome of women involved in minor trauma during pregnancy. We suggest that flow cytometry should not be routinely assessed in pregnant women involved in minor trauma.

The effect of hemorrhage control adjuncts on outcome in severe pelvic fracture: A multi-institutional study.

BACKGROUND: Hemodynamically unstable patients with severe pelvic fracture are a significant challenge to trauma surgeons and have high mortality. Significant variability across institutions in hemorrhage control adjuncts used to quell pelvic bleeding has been demonstrated. However, the effect of these methods on time to definitive bleeding control, type of resuscitation given, and outcomes remains unknown. We sought to elucidate those effects. METHODS: This was a multicenter retrospective review of severe pelvic fracture patients in shock between 2011 and 2016. Shock was defined as systolic blood pressure less than 90 mm Hg, heart rate greater than 120 beats per minute, or base deficit less than -5. Definitive bleeding control was defined as time to surgical control in the operating room or embolization by interventional radiology. Significance level was at p less than 0.05. RESULTS: A total of 279 severe pelvic fracture patients with shock on admission from 12 trauma centers were included. The cohort was primarily male (62%) with median (interquartile range) age of 40 years (28-54 years), Injury Severity Score of 38 (29-50), and Glasgow Coma Scale score of 13 (3-15). Overall mortality was 32%. The most common adjunct used was pelvic binder (50%) followed by no adjunct (30.5%); least common was resuscitative balloon occlusion of the aorta (REBOA) (2.5%). Preperitoneal packing alone and REBOA alone/with other adjunct(s) resulted in the fastest times to operating room/interventional radiology but also had the highest blood utilization and mortality rates. Resuscitative balloon occlusion of the aorta was most often used along with pelvic binder (6 of 13; 46%). CONCLUSION: Marked variation in management of severe pelvic fracture patients in shock indicates the need for a standardized approach to maximize outcomes and minimize transfusion requirements. The use of preperitoneal packing and/or REBOA yielded fastest times to definitive bleeding control. However, REBOA continues to be infrequently used. Future prospective analysis of this combination needs further validation in patients with severe pelvic hemorrhage. LEVEL OF EVIDENCE: Therapeutic study, level IV.

Expression of a recombinant, 4'-Phosphopantetheinylated, active M. tuberculosis fatty acid synthase I in E. coli.

BACKGROUND: Fatty acid synthase 1 (FAS I) from Mycobacterium tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4'-Phosphopantetheine (4'-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4'-PP transferase, termed acyl carrier protein synthase (AcpS). METHODS: In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid. We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. RESULTS: Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. CONCLUSION: Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structure-function analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

Terrorist Stabbings-Distinctive Characteristics and How to Prepare for Them.

BACKGROUND: The number of terror attack incidents is on the increase worldwide. The knife is one of the weapons most commonly used among terrorists. Appropriate preparation in trauma units for coping with the increasing numbers of terrorist-inflicted stabbings is different from the preparation suitable for civilian stabbings. Therapeutic and logistic guidelines need to be adjusted to accommodate those differences. OBJECTIVES: Characterize the unique injuries related to terrorist stabbing, and suggest preparedness actions. METHODS: Retrospective data on all terrorist-inflicted stabbing incidents between September 2015 and May 2016 were retrieved from the database of the national Israeli emergency medical services and from the Israeli Defense Forces Medical Corps records. RESULTS: There were a total of 414 civilian victims (34 fatalities) of terror incidents. Of these, 161 involved stabbings during 106 separate incidents. There was more than 1 stab wound per patient in approximately 60% of cases, and more than 1 victim in approximately 40% of cases. Unlike civilian stabbings, terrorist stabbings were characterized by more commonly occurring to the upper part of the body, being executed by large knives with high force, and involving multiple and more severe injuries. CONCLUSION: There is a clear distinction between the characteristics of wounds resulting from civilian stabbings and those incurred by acts of terror. Terrorists intend to injure as many random victims as possible, and trauma units need to be prepared to cope with the simultaneous admission of multiple patients with penetrating and often life-threatening knife wounds.

AcpM, the meromycolate extension acyl carrier protein of Mycobacterium tuberculosis, is activated by the 4'-phosphopantetheinyl transferase PptT, a potential target of the multistep mycolic acid biosynthesis.

Modification of acyl carrier proteins (ACP) or domains by the covalent binding of a 4'-phosphopantetheine (4'-PP) moiety is a fundamental condition for activation of fatty acid synthases (FASes) and polyketide synthases (PKSes). Binding of 4'-PP is mediated by 4' phosphopantetheinyl transfersases (PPTases). Mycobacterium tuberculosis (Mtb) possesses two essential PPTases: acyl carrier protein synthase (Mtb AcpS), which activates the multidomain fatty acid synthase I (FAS I), and Mtb PptT, an Sfp-type broad spectrum PPTase that activates PKSes. To date, it has not been determined which of the two Mtb PPTases, AcpS or PptT, activates the meromycolate extension ACP, Mtb AcpM, en route to the production of mycolic acids, the main components of the mycobacterial cell wall. In this study, we tested the enzymatic activation of a highly purified Mtb apo-AcpM to Mtb holo-AcpM by either Mtb PptT or Mtb AcpS. By using SDS-PAGE band shift assay and mass spectrometry analysis, we found that Mtb PptT is the PPTase that activates Mtb AcpM. We measured the catalytic activity of Mtb PptT toward CoA, using an activation assay of a blue pigment synthase, BpsA (a nonribosomal peptide synthase, NRPS). BpsA activation by Mtb PptT was inhibited by Mtb apo-AcpM through competition for CoA, in accord with Mtb AcpM activation. A structural model of the putative interaction between Mtb PptT and Mtb AcpM suggests that both hydrophobic and electrostatic interactions stabilize this complex. To conclude, activation of Mtb AcpM by Mtb PptT reveals a potential target of the multistep mycolic acid biosynthesis.

Abdominal cocoon: a potential pitfall in patients with ovarian carcinoma.

Background. Abdominal cocoon, or sclerosing encapsulating peritonitis, is a rare condition characterized by partial or total encasement of small bowel and mesentery by a thick fibrocollagenous sack that looks like a cocoon. Within the sack, bowel loops are drawn together causing intestinal obstruction.Case presentation. We report on a 57-year-old female patient who developed a very unusual complication of ovarian cancer: abdominal cocoon formation.Conclusions. This report highlights the need for a timely diagnosis of sclerosing encapsulating peritonitis in cancer patients.

Structure-function analysis of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis.

We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 A resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs.

Observation of calcium-dependent unidirectional rotational motion in recombinant photosynthetic F1-ATPase molecules.

ATP hydrolysis and synthesis by the F(0)F(1)-ATP synthase are coupled to proton translocation across the membrane in the presence of magnesium. Calcium is known, however, to disrupt this coupling in the photosynthetic enzyme in a unique way: it does not support ATP synthesis, and CaATP hydrolysis is decoupled from any proton translocation, but the membrane does not become leaky to protons. Understanding the molecular basis of these calcium-dependent effects can shed light on the as yet unclear mechanism of coupling between proton transport and rotational catalysis. We show here, using an actin filament gamma-rotation assay, that CaATP is capable of sustaining rotational motion in a highly active hybrid photosynthetic F(1)-ATPase consisting of alpha and beta subunits from Rhodospirillum rubrum and gamma subunit from spinach chloroplasts (alpha(R)(3)beta(R)(3)gamma(C)). The rotation was found to be similar to that induced by MgATP in Escherichia coli F(1)-ATPase molecules. Our results suggest a possible long range pathway that enables the bound CaATP to induce full rotational motion of gamma but might block transmission of this rotational motion into proton translocation by the F(0) part of the ATP synthase.