Mass spectrometry-based methods for characterizing transient protein–protein interactions

The dynamic associations of transient protein–protein interactions (PPIs) are critical mediators of myriad biochemical processes. These specific, low-affinity interactions are often mediated by conserved amino acid sequences or short linear motifs (SLiMs) that interact with corresponding binding domains. The short-lived and dynamic nature of these interactions make their biophysical characterization a significant challenge. This review focuses on the development and future directions of mass spectrometry (MS)-based techniques for elucidating and characterizing SLiM-mediated PPIs. This includes the application of protein footprinting techniques to infer the location of SLiM binding sites and the growing role of native MS for direct observation of protein–SLiM interactions, highlighting their potential for the assessment of small molecule modulation of transient PPIs and the identification of interfacial SLiMs.</p

The spectrum and outcome of paediatric traumatic brain injury in KwaZulu-Natal Province, South Africa has not changed over the last two decades

Objectives. This retrospective review of a prospectively entered and maintained hybrid electronic trauma registry was intended to develop a comprehensive overview of traumatic brain injury (TBI) in children and adolescents and to compare it with previous audits from our local environment and from other developing world centres. All TBI patients admitted to hospital were included in this study. We reviewed the age, gender, outcomes, radiological findings and treatment of the patients.Methods. All patients aged ≤18 years old who were admitted by the Pietermaritzburg Metropolitan Trauma Service (PMTS) with TBI between December 2012 and December 2016 were included in this audit.Results. During the 4-year period under review, a total of 563 children and adolescents were treated for TBI by the PMTS. The median age was 6.4 years and 29% (n=165) were females. The mechanism of TBI was blunt trauma in 96% (n=544) of cases, with 4% (n=19) suffering penetrating trauma. The penetrating mechanisms included impalement by a cow horn and miscellaneous injuries due to saws, axes, barbed wire, spades, stones and knives. The blunt mechanisms included falls (n=102), assaults (n=108), collapse of a building (n=28), bicyclerelated injury (n=14), falling off a moving vehicle (n=280), motor vehicle accident (MVA; n=59), pedestrian vehicle accident (PVA; n=183) and animal-related injuries (n=8). There were 454 (80%) mild, 67 (12%) moderate and 42 (7%) severe cases of TBI. A total of 48 patients were admitted to the intensive care unit and 23 were admitted to the high care unit. Nine patients died. All the deaths were in the MVA and PVA group. The spectrum of TBI as diagnosed on computed tomography scans was nonspecific cerebral contusion (n=92), depressed skull fracture (n=70), sub-arachnoid haemorrhage (n=60), extradural haemorrhage (n=41), intracerebral haemorrhage (n=19), free air (n=19),subdural haemorrhage (n=13), intraventricular haemorrhage (n=9). A total of 62 (11%) patients required surgery.Conclusion. There is a significant burden of paediatric TBI in Pietermaritzburg. The majority of TBI was related to blunt trauma and assaults were very common. Although the short-term outcomes are good, the long-term consequences are poorly understood. Injury prevention programmes are needed to help reduce this burden of disease and a nationwide trauma registry is long overdue

Validation of the Simplified Motor Score in patients with traumatic brain injury at a major trauma centre in South Africa

Background. This study used data from a large prospectively entered database to assess the efficacy of the motor score (M score) component of the Glasgow Coma Scale (GCS) and the Simplified Motor Score (SMS) in predicting overall outcome in patients with traumatic brain injury (TBI).Objective. To safely and reliably simplify the scoring system used to assess level of consciousness of trauma patients in the acute setting.Methods. A retrospective observational review of the Pietermaritzburg Metropolitan Trauma Service hybrid electronic medical registry database was performed during the period January 2013 - December 2015. Patients were classified into three groups using their GCS as an injury severity score. These were mild TBI (GCS 13 - 15), moderate TBI (GCS 9 - 12) and severe TBI (GCS &lt;9). The Glasgow M score was specifically evaluated to determine the relationship between the individual motor component and patient outcome.Results. GCS scores and M scores were analysed in a total of 830 patients. There was a decline in survival rate when the M score on admission was ≤4. The decline was more significant when the M score was ≤3. Survival rates were 26.8% (11/41) for patients with an M score of 1, 63.6% (14/22) for those with a score of 2, 56.5% (13/23) for those with a score of 3, 80.0% (20/25) for those with a score of 4, and 95.5% (121/128) for those with a score of 5. Of 591 patients with an M score of 6, 580 (98.1%) survived. Mortality rose dramatically with declining SMS. This was highly significant. When the M score was plotted against mortality in 830 patients, there was a correct prediction in 769 cases (accuracy 92.7%, sensitivity 67.6%, specificity 95%). The area under the receiver operating characteristic (ROC) curve was 0.9037, with a standard deviation (area) of 0.0227. When comparing the SMS against mortality, the accuracy was 77.1%, the sensitivity 84.5% and the specificity 76.4%. The fitted ROC area was 0.891 and the empirical ROC area 0.86.Conclusion. The M score component of the GCS and the SMS accurately predict outcome in patients with TBI. In cases where the full GCS is difficult to assess, the M score and SMS can be used safely as a triage tool

Trauma electives in South Africa provide valuable training for international surgeons

Background. Trauma electives in South Africa (SA) are common and many foreign-based surgeons have undertaken such electives over the past 3 decades. Despite this, little academic attention has been paid to these electives, which remain largely informal and unstructured. This project aims to redress this deficit.Objectives. To investigate and document the extent of trauma clinical electives and to assess their impact on the careers of foreign surgeons who have undertaken such electives.Methods. A mixed methods-style questionnaire was compiled, which sought to document the demographics of surgeons undertaking an SA trauma clinical elective, the trauma clinical experience they had prior to the elective, as well as the volume of experience they acquired during the elective.Results. Sixty questionnaires were sent out and 21 were completed. There were 16 male and 5 female respondents. Only 17 had undertaken a formal trauma rotation before their elective in SA. The mean number of major resuscitations managed prior to rotating through surgery departments in SA was 15, and the mean number managed during a 12-month rotation in SA was 204. It would take each respondent 14 years in their country of origin to acquire an equivalent level of exposure to major resuscitation. During the year before their elective, each surgeon had been exposed to a mean number of the following: 0.5 gunshot wounds (GSWs), 2 stab wounds (SWs), 0.1 blast injuries and 19 road traffic accidents (RTAs). The equivalent mean number for their year in SA was 106 GSWs, 153 SWs, 4 blast injuries and 123 RTAs. The time necessary to achieve a similar level of exposure to their SA experience if they had remained in their country of origin was 213 years for GSWs, 73 years for SWs, 41 years for blast injuries and 7 years for RTAs. Compared with their SA elective, it would take each respondent 3 years to insert as many central venous lines, 9 years to perform the same number of tube thoracostomies, 9 years to manage as many surgical airways, 18 years to explore as many SWs of the neck and 93 years to explore as many GSWs of the neck. Furthermore, it would take 33 years to see and perform as many laparotomies for SWs to the abdomen, 374 years to perform an equivalent number of GSWs to the abdomen and 34 years of experience to perform as many damage-control laparotomies in their countries of origin. In terms of vascular trauma, it would take 23 years to see as many vascular injuries secondary to SWs and 77 years to see an equivalent number of vascular injuries secondary to GSWs.Conclusions. A trauma clinical elective in SA provides an unparalleled exposure to almost all forms of trauma in conjunction with a welldeveloped academic support programme. Formalising these trauma electives might allow for the development of exchange programmes for SA trainees who wish to acquire international exposure to advanced general surgical training

Defining the need for surgical intervention following a snakebite still relies heavily on clinical assessment: The experience in Pietermaritzburg, South Africa

Background. This audit of snakebites was undertaken to document our experience with snakebite in the western part of KwaZulu-Natal (KZN) Province, South Africa (SA).Objective. To document our experience with snakebite in the western part of KZN, and to interrogate the data on patients who required some form of surgical intervention.Methods. A retrospective study was undertaken at the Pietermaritzburg Metropolitan Trauma Service, Pietermaritzburg, SA. The Hybrid Electronic Medical Registry was reviewed for the 5-year period January 2012 - December 2016. All patients admitted to the service for management of snakebite were included.Results. The offending snake is rarely identified, and the syndromic approach is now the mainstay of management. Most envenomations seen during the study period were cytotoxic, presenting with painful progressive swelling (PPS). We did not see any purely neurotoxic or haemotoxic envenomations. Antivenom is required for a subset of patients. The indications are essentially PPS that increases by &gt;15 cm over an hour, PPS up to the elbow or knee after 4 hours, PPS of the whole limb after 8 hours, threatened airway, shortness of breath, associated clotting abnormalities and compartment syndrome. If no symptoms have manifested within 1 hour of a snakebite, clinically significant envenomation is unlikely to have occurred. Antivenom is associated with a high rate of anaphylaxis and should only be administered when absolutely indicated, preferably in a high-care setting under continuous monitoring. The need for surgery is less well defined. Urgent surgery is indicated for compartment syndrome of the limb, which is a potentially life- and limb-threatening condition. Its diagnosis is usually made clinically, but this is difficult in snakebites. Morbidity and cost increase dramatically once fasciotomy is required, as evidenced by much longer hospital stay. There is frequently a degree of cross-over between cytotoxicity and haemotoxicity in envenomations that require fasciotomy, which means that fasciotomy may result in catastrophic bleeding and should be preceded by the administration of antivenom, especially in patients with a low platelet count or a high international normalised ratio. Physiological and biochemical markers are unhelpful in assessing the need for fasciotomy. Objective methods include measurement of compartment pressures and ultrasound.Conclusion. The syndromic management of snakebite is effective and safe. There is a high incidence of anaphylactic reactions to antivenom, and its administration must be closely supervised. In our area we overwhelmingly see cytotoxic snakebites with PPS. Surgery is often needed, and we need to refine our algorithms in terms of deciding on surgery

Native Mass Spectrometry-Guided Screening Identifies Hit Fragments for HOP-HSP90 PPI Inhibition

Contemporary medicinal chemistry considers fragment‐based drug discovery (FBDD) and inhibition of protein‐protein interactions (PPI) as important means of expanding the volume of druggable chemical space. However, the ability to robustly identify valid fragments and PPI inhibitors is an enormous challenge, requiring the application of sensitive biophysical methodology. Accordingly, in this study, we exploited the speed and sensitivity of nanoelectrospray (nano‐ESI) native mass spectrometry to identify a small collection of fragments which bind to the TPR2AB domain of HOP. Follow‐up biophysical assessment of a small selection of binding fragments confirmed binding to the single TPR2A domain, and that this binding translated into PPI inhibitory activity between TPR2A and the HSP90 C‐terminal domain. An in‐silico assessment of binding fragments at the PPI interfacial region, provided valuable structural insight for future fragment elaboration strategies, including the identification of losartan as a weak, albeit dose‐dependent inhibitor of the target PPI

Bounded Model Checking of Concurrent Data Types on Relaxed Memory Models: A Case Study

Many multithreaded programs employ concurrent data types to safely share data among threads. However, highly-concurrent algorithms for even seemingly simple data types are difficult to implement correctly, especially when considering the relaxed memory ordering models commonly employed by today’s multiprocessors. The formal verification of such implementations is challenging as well because the high degree of concurrency leads to a large number of possible executions. In this case study, we develop a SAT-based bounded verification method and apply it to a representative example, a well-known two-lock concurrent queue algorithm. We first formulate a correctness criterion that specifically targets failures caused by concurrency; it demands that all concurrent executions be observationally equivalent to some serial execution. Next, we define a relaxed memory model that conservatively approximates several common shared-memory multiprocessors. Using commit point specifications, a suite of finite symbolic tests, a prototype encoder, and a standard SAT solver, we successfully identify two failures of a naive implementation that can be observed only under relaxed memory models. We eliminate these failures by inserting appropriate memory ordering fences into the code. The experiments confirm that our approach provides a valuable aid for desigining and implementing concurrent data types

Exclusion limits on the WIMP-nucleon cross-section from the Cryogenic Dark Matter Search

The Cryogenic Dark Matter Search (CDMS) employs low-temperature Ge and Si detectors to search for Weakly Interacting Massive Particles (WIMPs) via their elastic-scattering interactions with nuclei while discriminating against interactions of background particles. For recoil energies above 10 keV, events due to background photons are rejected with >99.9% efficiency, and surface events are rejected with >95% efficiency. The estimate of the background due to neutrons is based primarily on the observation of multiple-scatter events that should all be neutrons. Data selection is determined primarily by examining calibration data and vetoed events. Resulting efficiencies should be accurate to about 10%. Results of CDMS data from 1998 and 1999 with a relaxed fiducial-volume cut (resulting in 15.8 kg-days exposure on Ge) are consistent with an earlier analysis with a more restrictive fiducial-volume cut. Twenty-three WIMP candidate events are observed, but these events are consistent with a background from neutrons in all ways tested. Resulting limits on the spin-independent WIMP-nucleon elastic-scattering cross-section exclude unexplored parameter space for WIMPs with masses between 10-70 GeV c^{-2}. These limits border, but do not exclude, parameter space allowed by supersymmetry models and accelerator constraints. Results are compatible with some regions reported as allowed at 3-sigma by the annual-modulation measurement of the DAMA collaboration. However, under the assumptions of standard WIMP interactions and a standard halo, the results are incompatible with the DAMA most likely value at >99.9% CL, and are incompatible with the model-independent annual-modulation signal of DAMA at 99.99% CL in the asymptotic limit.Comment: 40 pages, 49 figures (4 in color), submitted to Phys. Rev. D; v.2:clarified conclusions, added content and references based on referee's and readers' comments; v.3: clarified introductory sections, added figure based on referee's comment