ROS Consumers or Producers? Interpreting Transcriptomic Data by AlphaFold Modeling Provides Insights into Class III Peroxidase Functions in Response to Biotic and Abiotic Stresses.

Participating in both biotic and abiotic stress responses, plant-specific class III peroxidases (PERs) show promise as candidates for crop improvement. The multigenic PER family is known to take part in diverse functions, such as lignin formation and defense against pathogens. Traditionally linked to hydrogen peroxide (H2O2) consumption, PERs can also produce reactive oxygen species (ROS), essential in tissue development, pathogen defense and stress signaling. The amino acid sequences of both orthologues and paralogues of PERs are highly conserved, but discovering correlations between sequence differences and their functional diversity has proven difficult. By combining meta-analysis of transcriptomic data and sequence alignments, we discovered a correlation between three key amino acid positions and gene expression in response to biotic and abiotic stresses. Phylogenetic analysis revealed evolutionary pressure on these amino acids toward stress responsiveness. Using AlphaFold modeling, we found unique interdomain and protein-heme interactions involving those key amino acids in stress-induced PERs. Plausibly, these structural interactions may act as "gate keepers" by preventing larger substrates from accessing the heme and thereby shifting PER function from consumption to the production of ROS.

Association Between Profound Shock Signs and Peripheral Intravenous Access Success Rates in Trauma Patients in the Prehospital Scenario: A Retrospective Study.

BACKGROUND: Hemorrhage is the leading cause of preventable death in trauma patients, and establishment of intravenous (IV) access is essential for volume resuscitation, a key component in the treatment of hemorrhagic shock. IV access among patients in shock is generally considered more challenging, although data to support this notion are lacking. METHODS: In this retrospective registry-based study, data were collected from the Israeli Defense Forces Trauma Registry (IDF-TR) regarding all prehospital trauma patients treated by IDF medical forces between January 2020 and April 2022, for whom IV access was attempted. Patients younger than 16 years, nonurgent patients, and patients with no detectable heart rate or blood pressure were excluded. Profound shock was defined as a heart rate >130 or a systolic blood pressure <90 mm Hg, and comparisons were made between patients with profound shock and those not exhibiting such signs. The primary outcome was the number of attempts required for first IV access success, which was regarded as an ordinal categorical variable: 1, 2, 3 and higher and ultimate failure. A multivariable ordinal logistic regression was performed to adjust for potential confounders. Patients' sex, age, mechanism of injury and best consciousness level, as well as type of event (military/nonmilitary), and the presence of multiple patients were included in the ordinal logistic regression multivariable analysis model based on previous publications. RESULTS: Five hundred thirty-seven patients were included, 15.7% of whom were recorded as having signs of profound shock. Peripheral IV access establishment first attempt success rates were higher in the nonshock group, and there was a lower rate of unsuccessful attempts in this group (80.8% vs 67.8% for the first attempt, 9.4% vs 16.7% for the second attempt, 3.8% vs 5.6% for the third and further attempts, and 6% vs 10% unsuccessful attempts, P = .04). In the univariable analysis, profound shock was associated with requirement for an increased number of IV attempts (odds ratio [OR], 1.94; confidence interval [CI], 1.17-3.15). The ordinal logistic regression multivariable analysis demonstrated that profound shock was associated with worse results regarding primary outcome (adjusted odds ratio [AOR], 1.84; CI, 1.07-3.10). CONCLUSIONS: The presence of profound shock in trauma patients in the prehospital scenario is associated with an increased number of attempts required for IV access establishment.

A feasibility study using sodium alginate injection for penetrating abdominal trauma in a swine model.

Penetrating abdominal injury is a major cause of death in trauma. Sodium alginate hydrogel, a hemostatic agent, offers a platform for targeting both mechanical and biological injuries. The current study assessed the effect of Very Low Viscosity (high) G (VLVG) alginate following abdominal trauma in a swine model of penetrating abdominal injury. Seven anesthetized pigs were instrumented with invasive monitoring catheters and abdominal trauma was introduced by laparoscopic hepatectomy. Ten minutes after the induction of hypovolemic shock, three animals were intra-abdominally administered with VLVG alginate (study group) and four animals with saline (control group). During 8 h of continuous monitoring, various hemodynamic and biochemical variables were measured and liver biopsies for histological evaluation were taken. Hemodynamically, VLVG alginate-treated animals were more stable than controls, as reflected by their lower heart rate and higher blood pressure (p < 0.05 for both). They also had lower levels of liver enzymes and lactate, and less histopathological damage. We show that VLVG alginate might be a promising new agent for reducing penetrating intra-abdominal injury, with hemostatic and biocompatibility efficiency, and tissue preserving properties. Future effort of integrating it with a dispersal device may turn it into a valuable pre-hospital emergency tool to improve survival of trauma casualties.

Traumatic cerebral dural sinus vein thrombosis/stenosis in pediatric patients-is anticoagulation necessary?

INTRODUCTION: Cerebral dural vein thrombosis/stenosis (CDVT/S) is a condition that affects the venous drainage of the brain. Risk factors and causes associated with CDVT/S include systemic risk factors that cause hypercoagulability, or local factors such as head trauma. While consensus is that non-traumatic sinus vein thrombosis should be treated with anticoagulation therapy, treatment of patients with TBI-induced CDVT is not yet established. METHODS: Retrospective review of clinical data of pediatric patients presented to our medical center from July 2017 to August 2020. Inclusion criteria were age, birth to 18 years, admission due to head trauma, head CT scan with positive traumatic findings, and follow-up in our clinic. Exclusion criteria were a normal head CT on admission and failure to follow-up. Data regarding demographics, clinical presentation, imaging findings, treatment, and status on follow-up were recorded. Study protocol was approved by our institutional ethics committee. RESULTS: One hundred sixty-two patients were enrolled. Falling accident occurred in 90.1%, a minority suffered from direct head trauma or gunshot wound. Of the patients, 95.1% suffered from mild TBI. Forty-two percent suffered from an associated intracranial injury. Fourteen cases with CDVT were included in the cohort. Linear fractures were significantly correlated with CDVT. Additionally, occipital/suboccipital fractures, associated intracranial injury, and proximity of injury to the sinus were correlated with CDVT. From this group, 12 were treated conservatively; one patient was treated surgically due to EDH. All patients with CDVT were neurologically intact at discharge. Only one patient was treated with therapeutic dose of LMWH. A total of 86.7% of patients with CDVT who were treated conservatively had full recanalization on follow-up imaging. Four patients had CDVS; all were neurologically intact at admission and discharge, and all were treated conservatively and had full recanalization on follow-up. DISCUSSION: Treatment with ACT is established in pediatric CDVT but not in the sub-group of TBI. While ACT prevents progression of thrombosis, it might cause worsening of extra-axial hemorrhage. In our study, no clinical deterioration was noted with expectant management; thus, we present an algorithm for diagnosis and treatment of trauma-induced CDVT/S in children with frequent clinical and radiologic imaging while avoiding anticoagulation. CONCLUSION: In most cases, anticoagulation therapy is not necessary in traumatic CDVT/S. Initial expectant management in children is safe. However, each case should be evaluated individually and further studies should be performed.

Outcome of Peripartum Anesthesia in Women with Valvular Disease.

OBJECTIVE: Maternal heart disease is one of the major causes for mortality among parturients. In our study, we surveyed 220 patients with different valvular disorders who gave birth in our medical center in the years 2012-2018. The aim of this study was to characterize various valvular pathologies and compare the results of different anesthetic approaches. METHODS: In this retrospective study, the computerized system and file archive were searched for maternal valvular pathologies according to the International Classification of Diseases, Ninth Revision (ICD-9). The women included in the study were defined as American Society of Anesthesiology (ASA)-II or more, who suffer from valvular heart disease. RESULTS: The most common pathology was mitral valve regurgitation (57.73% of all cardiac patients). Most women were defined as having mild insufficiency, and 82.68% had normal vaginal delivery. In 17.3% of patients who had cesarean section, the main type of anesthesia was neuraxial anesthesia (95.45%). The second most common pathology was tricuspid valve regurgitation (22.73%). Most patients (78%) had normal vaginal delivery, and epidural analgesia was used in 64.1%. A minority of cardiac patients in our study were patients with stenotic heart diseases, such as aortic stenosis, mitral stenosis and pulmonic stenosis (8.18%, 4.55%, and 1.36%, respectively). No complications were observed in the peripartum period. CONCLUSION: The use of regional anesthesia is recommended for all valvular pathologies without exception, as we observed no cases in which the severity of cardiac condition had not allowed the use of various types of regional anesthesia, for surgery or vaginal delivery.

Ion mobility-mass spectrometry reveals the influence of subunit packing and charge on the dissociation of multiprotein complexes.

The composition, stoichiometry, and organization of protein complexes can be determined by collision-induced dissociation (CID) coupled to tandem mass spectrometry (MS/MS). The increased use of this approach in structural biology prompts a better understanding of the dissociation mechanism(s). Here we report a detailed investigation of the CID of two dodecameric, heat-stable and toroidally shaped complexes: heat shock protein 16.9 (HSP16.9) and stable protein 1 (SP-1). While HSP16.9 dissociates by sequential loss of unfolded monomers, SP-1 ejects not only monomers, but also its building blocks (dimers), and multiples thereof (tetramers and hexamers). Unexpectedly, the dissociation of SP-1 is strongly charge-dependent: loss of the building blocks increases with higher charge states of this complex. By combining MS/MS with ion mobility (IM-MS/MS), we have monitored the unfolding and dissociation events for these complexes in the gas phase. For HSP16.9 unfolding occurs at lower energies than the ejection of subunits, whereas for SP-1 unfolding and dissociation take place simultaneously. We consider these results in the light of the structural organization of HSP16.9 and SP-1 and hypothesize that SP-1 is unable to unfold extensively due to its particular quaternary structure and unusually high charge density. This investigation increases our understanding of the factors governing the CID of protein complexes and moves us closer to the goal of obtaining structural information on subunit interactions and packing from gas-phase experiments.

Integrating ion mobility mass spectrometry with molecular modelling to determine the architecture of multiprotein complexes.

Current challenges in the field of structural genomics point to the need for new tools and technologies for obtaining structures of macromolecular protein complexes. Here, we present an integrative computational method that uses molecular modelling, ion mobility-mass spectrometry (IM-MS) and incomplete atomic structures, usually from X-ray crystallography, to generate models of the subunit architecture of protein complexes. We begin by analyzing protein complexes using IM-MS, and by taking measurements of both intact complexes and sub-complexes that are generated in solution. We then examine available high resolution structural data and use a suite of computational methods to account for missing residues at the subunit and/or domain level. High-order complexes and sub-complexes are then constructed that conform to distance and connectivity constraints imposed by IM-MS data. We illustrate our method by applying it to multimeric protein complexes within the Escherichia coli replisome: the sliding clamp, (beta2), the gamma complex (gamma3deltadelta'), the DnaB helicase (DnaB6) and the Single-Stranded Binding Protein (SSB4).

The mechanical properties of PCNA: implications for the loading and function of a DNA sliding clamp.

Sliding clamps are toroidal proteins that encircle DNA and act as mobile platforms for DNA replication and repair machinery. To be loaded onto DNA, the eukaryotic sliding clamp Proliferating Cell Nuclear Antigen (PCNA) must be splayed open at one of the subunit-subunit interfaces by the ATP-dependent clamp loader, Replication Factor C, whose clamp-interacting sites form a right-handed spiral. Earlier molecular dynamics (MD) studies suggested that when PCNA opens, it preferentially adopts a right-handed spiral to match the spiral of the clamp loader. Here, analysis of considerably longer MD simulations shows that although the opened form of PCNA can achieve conformations matching the helical pitch of Replication Factor C, it is not biased toward a right-handed spiral structure. A coarse-grained elastic model was also built; its strong correspondence to the all-atom MD simulations of PCNA suggests that the behavior of the open clamp is primarily due to elastic deformation governed by the topology of the clamp domains. The elastic model was further used to construct the energy landscape of the opened PCNA clamp, including conformations that would allow binding to the clamp loader and loading onto double-stranded DNA. A picture of PCNA emerges of a rather flexible protein that, once opened, is mechanically compliant in the clamp opening process.

A single subunit directs the assembly of the Escherichia coli DNA sliding clamp loader.

Multi-protein clamp loader complexes are required to load sliding clamps onto DNA. In Escherichia coli the clamp loader contains three DnaX (tau/gamma) proteins, delta, and delta', which together form an asymmetric pentameric ring that also interacts with psichi. Here we used mass spectrometry to examine the assembly and dynamics of the clamp loader complex. We find that gamma exists exclusively as a stable homotetramer, while tau is in a monomer-dimer-trimer-tetramer equilibrium. delta' plays a direct role in the assembly as a tau/gamma oligomer breaker, thereby facilitating incorporation of lower oligomers. With delta', both delta and psichi stabilize the trimeric form of DnaX, thus completing the assembly. When tau and gamma are present simultaneously, mimicking the situation in vivo, subunit exchange between tau and gamma tetramers occurs rapidly to form heterocomplexes but is retarded when deltadelta' is present. The implications for intracellular assembly of the DNA polymerase III holoenzyme are discussed.

Identification of a thermo-regulated glutamine-binding protein from Yersinia pestis.

Here we present modeling and NMR spectroscopic evidence that the function of a Yersinia pestis pMT1 plasmid protein, designated as orf38, is most likely a glutamine binding protein. The modeling was homology-based at a very low level of sequence identity ( approximately 16%) and involved structural comparison of multiple templates, as well as template-substrate interaction analyses. Transferred nuclear Overhauser and saturation transfer difference experiments were used to characterize the binding of sugars and amino acids to orf38. The identification and characterization of an unknown protein function using the strategy presented here has applicability to a variety of research areas, including functional genomics and proteomics efforts.

Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3.

The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome.

Motion of a DNA sliding clamp observed by single molecule fluorescence spectroscopy.

DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III beta subunit or beta clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the beta clamp diffusing along DNA is on the order of 10(-14) m(2)/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the beta clamp remains at the 3' end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.

Correlation spectroscopy of minor fluorescent species: signal purification and distribution analysis.

We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable signal from free sliding clamp and free DNA. When multiple fluorescent species contribute to a correlation signal, it is difficult or impossible to distinguish between contributions from individual species. As a remedy, we introduce "purified FCS", which uses single molecule burst analysis to select a species of interest and extract the correlation signal for further analysis. We show that by expanding the correlation region around a burst, the correlated signal is retained and the functional forms of FCS fitting equations remain valid. We demonstrate the use of purified FCS in experiments with DNA sliding clamps. We also introduce "single-molecule FCS", which obtains diffusion time estimates for each burst using expanded correlation regions. By monitoring the detachment of weakly-bound 30-mer DNA oligomers from a single-stranded DNA plasmid, we show that single-molecule FCS can distinguish between bursts from species that differ by a factor of 5 in diffusion constant.

Identification of 2-nonynoic acid, a cosmetic component, as a potential trigger of primary biliary cirrhosis.

Antimitochondrial antibodies (AMA) are unique among autoimmune serologic reactants because of their extremely high association with the index disease primary biliary cirrhosis (PBC). This autoantibody response is specifically directed only to the lipoyl domain of the mitochondrial 2-oxo-acid dehydrogenase complexes, which prompted us to search for environmental mimotopes in the form of xenobiotics and led to our identification of 2-octynoic acid as a high-affinity reactant for AMA. To focus on the chemical characteristics requisite for binding of AMA to the xenobiotic-modified self-peptide, quantitative structure-activity relationship (QSAR) studies were performed using a panel of alkynoic compounds, including examination of the length of the carbon chain and the location of the triple bond in the identified mimotope. Analyses of octynamides that varied in the position of the triple bond demonstrated that only the 2-octynamide reacted strongly with PBC sera. Furthermore, among 2-alkynamides with varying carbon chain length, 2-octyn-, 2-nonyn- (particularly) and 2-decynamide exhibited the highest reactivity. Thus, an optimal chemical structure of the xenobiotically modified epitope recognized by AMA-positive PBC sera is provided by 2-nonynoic acid. The methyl ester of this compound is ranked 2,324th out of 12,945 compounds to which there is occupational exposure, with an 80% female prevalence due to its use in cosmetic products. Our findings illustrate an unusual polyreactivity of anti-PDC-E2 and support the idea of epitope mimicry in the genesis of this autoantibody and perhaps of PBC itself.

A non-cleavable UmuD variant that acts as a UmuD' mimic.

UmuD(2) cleaves and removes its N-terminal 24 amino acids to form UmuD'(2), which activates UmuC for its role in UV-induced mutagenesis in Escherichia coli. Cells with a non-cleavable UmuD exhibit essentially no UV-induced mutagenesis and are hypersensitive to killing by UV light. UmuD binds to the beta processivity clamp ("beta") of the replicative DNA polymerase, pol III. A possible beta-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with beta. We performed alanine-scanning mutagenesis of this motif ((14)TFPLF(18)) in UmuD and found that it has a moderate influence on UV-induced mutagenesis but is required for the cold-sensitive phenotype caused by elevated levels of wild-type UmuD and UmuC. Surprisingly, the wild-type and the beta-binding motif variant bind to beta with similar K(d) values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in beta is in strikingly different environments in the presence of the wild-type versus the variant UmuD proteins, suggesting a distinct change in some aspect of the interaction with little change in its strength. Despite the fact that this novel UmuD variant is non-cleavable, we find that cells harboring it display phenotypes more consistent with the cleaved form UmuD', such as resistance to killing by UV light and failure to exhibit the cold-sensitive phenotype. Cross-linking and chemical modification experiments indicate that the N-terminal arms of the UmuD variant are less likely to be bound to the globular domain than those of the wild-type, which may be the mechanism by which this UmuD variant acts as a UmuD' mimic.

Two processivity clamp interactions differentially alter the dual activities of UmuC.

DNA polymerases of the Y family promote survival by their ability to synthesize past lesions in the DNA template. One Escherichia coli member of this family, DNA pol V (UmuC), which is primarily responsible for UV-induced and chemically induced mutagenesis, possesses a canonical beta processivity clamp-binding motif. A detailed analysis of this motif in DNA pol V (UmuC) showed that mutation of only two residues in UmuC is sufficient to result in a loss of UV-induced mutagenesis. Increased levels of wild-type beta can partially rescue this loss of mutagenesis. Alterations in this motif of UmuC also cause loss of the cold-sensitive and beta-dependent synthetic lethal phenotypes associated with increased levels of UmuD and UmuC that are thought to represent an exaggeration of a DNA damage checkpoint. By designing compensatory mutations in the cleft between domains II and III in beta, we restored UV-induced mutagenesis by a UmuC beta-binding motif variant. A recent co-crystal structure of the 'little finger' domain of E. coli pol IV (DinB) with beta suggests that, in addition to the canonical beta-binding motif, a second site of pol IV ((303)VWP(305)) interacts with beta at the outer rim of the dimer interface. Mutational analysis of the corresponding motif in UmuC showed that it is dispensable for induced mutagenesis, but that alterations in this motif result in loss of the cold-sensitive phenotype. These two beta interaction sites of UmuC affect the dual functions of UmuC differentially and indicate subtle and sophisticated polymerase management by the beta clamp.

DNA sliding clamps: just the right twist to load onto DNA.

Two recent papers illuminate a key step in DNA sliding clamp loading: one reveals the structure of the PCNA clamp wrapped around DNA--still open from being loaded--while the other finds that the clamp may assist this process by forming a right-handed helix upon opening.

Domain mapping of the Rad51 paralog protein complexes.

The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and Rad51C-Xrcc3 (CX3). Based on the recent Pyrococcus furiosus Rad51 structure, we have used homology modeling to design deletion mutants of the Rad51 paralogs. The models of the human Rad51B, Rad51C, Xrcc3 and murine Rad51D (mRad51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region. Using yeast two-hybrid and co-immunoprecipitation techniques, we have demonstrated that a fragment of Rad51B containing amino acid residues 1-75 interacts with the C-terminus and linker of Rad51C, residues 79-376, and this region of Rad51C also interacts with mRad51D and Xrcc3. We have also determined that the N-terminal domain of mRad51D, residues 4-77, binds to Xrcc2 while the C-terminal domain of mRad51D, residues 77-328, binds Rad51C. By this, we have identified the binding domains of the BCDX2 and CX3 complexes to further characterize the interaction of these proteins and propose a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes.

Molecular interactions of human Exo1 with DNA.

Human Exo1 is a member of the RAD2 nuclease family with roles in replication, repair and recombination. Despite sharing significant amino acid sequence homology, the RAD2 proteins exhibit disparate nuclease properties and biological functions. In order to identify elements that dictate substrate selectivity within the RAD2 family, we sought to identify residues key to Exo1 nuclease activity and to characterize the molecular details of the human Exo1-DNA interaction. Site-specific mutagenesis studies demonstrate that amino acids D78, D173 and D225 are critical for Exo1 nuclease function. In addition, we show that the chemical nature of the 5'-terminus has a major impact on Exo1 nuclease efficiency, with a 5'-phosphate group stimulating degradation 10-fold and a 5'-biotin inhibiting degradation 10-fold (relative to a 5'-hydroxyl moiety). An abasic lesion located within a substrate DNA strand impedes Exo1 nucleolytic degradation, and a 5'-terminal abasic residue reduces nuclease efficiency 2-fold. Hydroxyl radical footprinting indicates that Exo1 binds predominantly along the minor groove of flap DNA, downstream of the junction. As will be discussed, our results favor the notion that the single-stranded DNA structure is pinched by the helical arch of the protein and not threaded through this key recognition loop. Furthermore, our studies indicate that significant, presumably biologically relevant, differences exist between the active site dynamics of Exo1 and Fen1.