Robotic sample changers for macromolecular X-ray crystallography and biological small-angle X-ray scattering at the National Synchrotron Light Source II. Corrigendum.

A correction in the paper by Lazo et al. [(2021). J. Synchrotron Rad. 28, 1649-1661] is made.

AMX - the highly automated macromolecular crystallography (17-ID-1) beamline at the NSLS-II.

The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 × 1012 photons s-1), micro-focus (7 µm × 5 µm), low-divergence (1 mrad × 0.35 mrad) energy-tunable (5-18 keV) beamline at the NSLS-II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17-ID with the FMX beamline, the frontier micro-focus macromolecular crystallography beamline. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro-focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17-ID-1 as well as some plans for the near future are presented.

Robotic sample changers for macromolecular X-ray crystallography and biological small-angle X-ray scattering at the National Synchrotron Light Source II.

Here we present two robotic sample changers integrated into the experimental stations for the macromolecular crystallography (MX) beamlines AMX and FMX, and the biological small-angle scattering (bioSAXS) beamline LiX. They enable fully automated unattended data collection and remote access to the beamlines. The system designs incorporate high-throughput, versatility, high-capacity, resource sharing and robustness. All systems are centered around a six-axis industrial robotic arm coupled with a force torque sensor and in-house end effectors (grippers). They have the same software architecture and the facility standard EPICS-based BEAST alarm system. The MX system is compatible with SPINE bases and Unipucks. It comprises a liquid nitrogen dewar holding 384 samples (24 Unipucks) and a stay-cold gripper, and utilizes machine vision software to track the sample during operations and to calculate the final mount position on the goniometer. The bioSAXS system has an in-house engineered sample storage unit that can hold up to 360 samples (20 sample holders) which keeps samples at a user-set temperature (277 K to 300 K). The MX systems were deployed in early 2017 and the bioSAXS system in early 2019.

FMX - the Frontier Microfocusing Macromolecular Crystallography Beamline at the National Synchrotron Light Source II.

Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser. 425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5-30 keV photon energy range and delivers a flux of 4.0 × 1012 photons s-1 at 1 Šinto a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 1012 photons s-1 at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods - serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25, 1362-1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238-246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.

High-speed raster-scanning synchrotron serial microcrystallography with a high-precision piezo-scanner.

The Frontier Microfocus Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II with its 1 µm beam size and photon flux of 3 × 1012 photons s-1 at a photon energy of 12.66 keV has reached unprecedented dose rates for a structural biology beamline. The high dose rate presents a great advantage for serial microcrystallography in cutting measurement time from hours to minutes. To provide the instrumentation basis for such measurements at the full flux of the FMX beamline, a high-speed, high-precision goniometer based on a unique XYZ piezo positioner has been designed and constructed. The piezo-based goniometer is able to achieve sub-100 nm raster-scanning precision at over 10 grid-linepairs s-1 frequency for fly scans of a 200 µm-wide raster. The performance of the scanner in both laboratory and serial crystallography measurements up to the maximum frame rate of 750 Hz of the Eiger 16M's 4M region-of-interest mode has been verified in this work. This unprecedented experimental speed significantly reduces serial-crystallography data collection time at synchrotrons, allowing utilization of the full brightness of the emerging synchrotron radiation facilities.

Community-acquired Methicillin-resistant Staphylococcus aureus Musculoskeletal Infections: Emerging Trends Over the Past Decade.

BACKGROUND: The emergence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) has altered the management of pediatric musculoskeletal infections. Yet, institution-to-institution differences in MRSA virulence may exist, suggesting a need to carefully examine local epidemiological characteristics. The purpose of this study was to compare MRSA and methicillin-sensitive S. aureus (MSSA) musculoskeletal infections with respect to prevalence and complexity of clinical care over the past decade at a single children's hospital. METHODS: We retrospectively reviewed a series of patients presenting to The Children's Hospital of Philadelphia with a diagnosis of osteomyelitis, septic arthritis, or both over a 10-year period. Inclusion criteria were S. aureus (SA) infections proven by positive culture of blood, bone, or joint aspirate. Exclusion criteria were non-SA infectious etiologies. Hospital-acquired infections were also not included to exclusively evaluate acute, community-acquired cases. Data related to hospital course, laboratory values, and number of surgical interventions were collected and compared between MRSA and MSSA cohorts. RESULTS: In our series of pediatric patients, we identified 148 cases of acute, community-acquired musculoskeletal SA infections (MRSA, n=37 and MSSA, n=111). The prevalence of MRSA musculoskeletal infections increased from 11.8% in 2001 to 2002 to 34.8% in 2009 to 2010. Compared with MSSA, MRSA infections resulted in higher presenting C-reactive protein levels (10.4 vs. 7.8 mg/L, P=0.04), longer inpatient stays (10 vs. 5 d, P<0.01), multiple surgical procedures (n>1) (38% vs. 14%, P<0.01), increased sequelae (27% vs. 6%, P<0.01), and more frequent admissions to the intensive care unit (16% vs. 3%, P<0.01). CONCLUSIONS: At our institution over the past decade, we found an approximate 3-fold rise in community-acquired pediatric MRSA musculoskeletal infections accompanied by an elevated risk for complications during inpatient management. Awareness of the epidemiological trends of MRSA within the local community may guide parental counseling and facilitate timely and accurate clinical diagnosis and treatment. LEVEL OF EVIDENCE: Level II-prognostic retrospective study.

Macromolecular crystallography beamline X25 at the NSLS.

Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource group. This mini-gap insertion-device beamline has seen constant upgrades for the last seven years in order to achieve mini-beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state-of-the-art facility for the scientific community.

Saphenous vein graft aneurysm with graft-enteric fistula after renal artery bypass.

A 65-year-old female presented with upper gastrointestinal hemorrhage thirty years following an aorta-to-right renal artery bypass constructed with saphenous vein. Upper endoscopy demonstrated a duodenal ulcer, and a CAT scan demonstrated aneurysmal degeneration of her renal artery bypass with duodenal impingement. Laparotomy demonstrated erosion of the aneurysm through the posterior wall of the duodenum; extra-anatomic renovascular reconstruction and primary duodenal repair was performed. Although aneurysmal degeneration of intraabdominal saphenous vein grafts is well described and rupture likewise reported, this report represents the first description of an intraabdominal autogenous vein graft aneurysm presenting with gastrointestinal erosion and fistula.

Balloon-expandable covered stent therapy of complex endovascular pathology.

The current study was designed to investigate our hypotheses that balloon-expandable covered stents display acceptable function over longitudinal follow-up in patients with complex vascular pathology and provide a suitable alternative for the treatment of recurrent in-stent restenosis. All stents were Atrium iCast, which is a balloon-mounted, polytetrafluoroethylene-covered stent with a 6F/7F delivery system. A retrospective review was performed of 49 patients with 66 stented lesions. Data were analyzed with life tables and t-tests. The most commonly treated vessels were the iliac (61%) and renal (24%) arteries. Indications for covered stent placement were unstable atheromatous lesions (50%), recurrent in-stent restenosis (24%), aneurysm (8%), aortic bifurcation reconstruction (7.5%), dissection (4.5%), endovascular aneurysm repair-related (4.5%), and stent fracture (1.5%). Patency was assessed by angiogram or duplex ultrasonography. The primary end point was patency and secondary end points were technical success and access-site complications. Mean follow-up was 13 months (range 1.5-25). The technical success rate was 97%. Unsuccessful outcomes were due to deployment error (n=1) and stent malpositioning (n=1). The cohort (n=64) 6- and 12-month primary patency rates were 96% and 84%, respectively. Twelve-month assisted primary patency was 98%. Iliac artery stents (n=38) had a primary patency of 97% at 6 months and 84% at 12 months with an assisted primary patency of 100% at 12 months. Renal artery stents (n=16) had a primary patency of 92% at 6 months and 72% at 12 months with an assisted primary patency of 92% at 6 and 12 months. Stents placed for recurrent in-stent restenosis (n=16) had a primary patency of 85%, assisted primary patency of 93%, and a 15% restenosis rate at 12 months. Specifically, stents placed for renal artery recurrent in-stent restenosis (n=10) had a primary patency of 73%, assisted primary patency of 82%, and a restenosis rate of 27%. The restenosis rate included two renal artery occlusions in patients noncompliant with clopidogrel use and resulted in ipsilateral kidney loss in both patients. In-stent peak systolic velocities decreased significantly (p<0.05) from preoperation to 12 months in iliac stents and to 18 months in renal stents. Ankle-brachial index increased significantly in iliac stents from preoperation (0.62+/-0.18) to 18 months (0.86+/-0.16). Successful exclusion of atheromatous lesions and aneurysm/dissection/endoleak was 100%. Access-site complications occurred in 6%: pseudoaneurysm (n=2), dissection (n=1), and bleeding (n=1). Balloon-expandable covered stents have an acceptable primary patency with an excellent assisted patency after salvage angioplasty. The clinical utility of this technology is broad for the treatment of aneurysms, extravasation, unstable atheromatous lesions, and recurrent in-stent restenosis.

Anterolateral Drawer Versus Anterior Drawer Test for Ankle Instability: A Biomechanical Model.

BACKGROUND: The addition of unconstrained internal rotation to the physical examination could allow for detection of more subtle degrees of ankle instability. We hypothesized that a simulated anterolateral drawer test allowing unconstrained internal rotation of the ankle would provoke greater displacement of the lateral talus in the mortise versus the anterior drawer test. METHODS: Ten cadaveric lower extremities were tested in a custom apparatus designed to reproduce the anterior drawer test and the anterolateral drawer test, in which the ankle was allowed to internally rotate about the intact deep deltoid ligament while being subluxed anteriorly. Specimens were tested intact and with anterior tibiofibular ligament sectioned. A differential variable reluctance transducer was used to measure lateral talar displacement with anterior forces of 25 and 50 N. RESULTS: No significant differences in talar displacement or ankle rotation were noted in intact specimens between the groups. Among sectioned specimens, significantly more talar displacement (25 N [6.5 ± 1.7 mm vs 3.8 ± 2.4 mm] and 50 N [8.7 ± 0.9 mm vs 4.5 ± 2.5 mm], P < .001) and ankle rotation (25 N [13.9 ± 8.0 degrees vs 0.0 ± 0.0 degrees] and 50 N [23.7 ± 5.8 degrees vs 0.0 ± 0.0 degrees], P < .001) were found in the anterolateral drawer versus anterior drawer group. CONCLUSION: In an ankle instability model, the anterolateral drawer test provoked almost twice the lateral talus displacement found with the anterior drawer test. CLINICAL RELEVANCE: Allowing internal rotation of the ankle while testing for ankle instability may allow the examiner to detect more subtle degrees of ankle instability.

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography.

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

Cholecystokinin (CCK) down regulates PGE2 and PGI2 release in inflamed Guinea pig gallbladder smooth muscle cell cultures.

This study examines the hypothesis that cholecystitis down-regulates Guinea pig gallbladder (GPGB) smooth muscle cholecystokinin (CCK)-stimulated prostaglandin (PG) release. Guinea pig gallbladder from Control and 48 h bile duct ligated (BDL) animals were placed in cell culture and grown to confluence. The cultures underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacyclin Synthase (PS), or were incubated with CCK at 10(-8)M or 10(-6)M with and without indomethacin for 1h and analyzed for release of 6-keto-PGF1alpha, PGE2 and TxB2 by EIA. BDL increased Guinea pig gallbladder cell culture basal PGE2 and PGI2 release which was in part due to increased COX-2 content. CCK incubation down-regulated BDL Guinea pig gallbladder cell culture release of 6-keto-PGF1alpha and PGE2 and down-regulated COX-2 content but did not alter the Control group. The decrease in CCK-mediated BDL cell Guinea pig gallbladder release may be an endogenous mechanism to limit physiologic derangements induced by increased endogenous gallbladder PG synthesis during early acute cholecystitis.

When exactly can carpal tunnel syndrome be considered work-related?

BACKGROUND: Carpal tunnel syndrome (CTS), compression of the median nerve at the wrist, is the most frequently encountered peripheral entrapment neuropathy. Whilst rates of all other work-related conditions have declined, the number of work-related musculoskeletal disorders (which include CTS) has not changed for the past 9 years in the USA. Median days off work are also highest for CTS: 27 compared to 20 for fractures and 18 for amputations. This results in enormous Workers Compensation and other costs to the community. Awareness of CTS as a disorder associated with repeated trauma at work is now so widespread amongst workers that many have diagnosed themselves before being medically assessed, often by means of the Internet. Surprisingly, however, a definite causal relationship has not yet been established for most occupations. Although the quality of research in this area is generally poor, CTS research studies are being used as the basis for acceptance of Workers Compensation claims, substantial expensive ergonomic workplace change and even workplace closures. The fact that the incidence of work-related musculoskeletal disorders has not changed despite these latter measures would suggest that a causal relationship is not proven and that some resources are being misdirected in CTS prevention and treatment. METHOD: A literature review of 64 articles on CTS was conducted. This included those articles most frequently cited as demonstrating the relationship between CTS and work. RESULTS: Primary risk factors in the development of CTS are: being a woman of menopausal age, obesity or lack of fitness, diabetes or having a family history of diabetes, osteoarthritis of the carpometacarpal joint of the thumb, smoking, and lifetime alcohol intake. In most cases, work acts as the 'last straw' in CTS causation. CONCLUSION: Except in the case of work that involves very cold temperatures (possibly in conjunction with load and repetition) such as butchery, work is less likely than demographic and disease-related variables to cause CTS. To label other types of work as having caused CTS, therefore, would result in inappropriate allocation of resources. It would also relieve individuals of the responsibility of addressing correctable lifestyle factors and treatable illnesses such as obesity, diabetes, smoking and increased alcohol intake which may have contributed to their CTS more that their work. This results in both avoidable long-term health effects and ongoing costs to the community.

Pharmacomechanical thrombolysis for phlegmasia cerulea dolens.

Phlegmasia cerulea dolens (PCD) is limb-threatening. Traditional treatments are very morbid. We examine the efficacy of percutaneous treatment of PCD. Between May 2005 and September 2008, we treated 21 limbs in 20 patients with lower extremity PCD who were candidates for thrombolysis. Diagnosis was by clinical examination and duplex ultrasound. Catheter access to the deep venous system was obtained through a popliteal vein. Therapy used pulse spray thrombolysis with tissue plasminogen activator (tPA). Infusion catheters and adjunctive percutaneous techniques were used as indicated. Postoperatively, patients were treated with systemic anticoagulation, compression hose, and interval follow-up. Limbs were graded according to the CEAP classification. Twenty patients (13 male) were treated with a mean age of 55.8 years. Nine patients had hypercoagulable states, four May Thurner syndrome, three a history of cancer, one postcolon resection, one acute myocardial infarction, and one postfemoral vein puncture. All patients had resolution of PCD without the need for open surgery. The initial tPA dose was 19.5 mg with pulse spray thrombolysis. Infusion catheters were required in 18 patients and used for 16.1 hours (range, 8 to 36 hours) until complete thrombolysis. Venous angioplasty was necessary in 14 patients with nine of these requiring venous stents. One patient required above-knee amputation despite successful treatment of her PCD. Mean follow-up was 10.7 months (range, 1 to 39 months). All patients demonstrated no or minimal residual thrombus and intact valvular function and a mean clinical CEAP score of 2.4. Percutaneous treatment of PCD produced excellent results with minimal morbidity.

External fixation of high-energy tibia fractures.

External fixation (EF) of tibia fractures has been associated with nonunions and malunions at our large pediatric trauma center. This study was designed to determine the successes and shortcomings of EF, especially with respect to maintenance of alignment and time to union. We believe that this will contribute to the limited amount of literature examining the complications associated with this treatment modality in the pediatric population. Thirty-one consecutive high-energy tibia fractures treated with EF over 4.5 years were analyzed. There were 22 boys and 9 girls (4-17 years old; mean, 11.9 years). Mean length of follow-up was 15 months. Of the 31 fractures analyzed, 19 were open fractures (12 closed, 3 grade I, 9 grade II, and 7 grade III). Of 30 fractures, 3 required skin graft, whereas 7 required fasciotomy. Mean duration of EF was 3.2 months. Mean time to union was 4.8 months. For complication rates, 4 of 30 had delayed union, 2 of 30 had nonunion, 8 of 30 had minor malunion, 3 of 30 had major malunion, 3 of 30 had leg length discrepancy, 8 of 30 had pin track infection, 3 of 30 had wound infection, 2 of 30 had osteomyelitis, and 4 of 30 required surgery for nonunion. Time to union differed between those aged 11 years or younger and those aged 12 years or older (means of 3.2 and 6.0 months, respectively; P = 0.001). Union time also differed between those with closed or grade I open fractures and those with grade II or III open fractures (3.9 and 5.7 months, respectively; P = 0.035). Leg length discrepancy rate differed between children aged 11 years or younger and those aged 12 years or older (3/13 and 0/18, respectively; P = 0.05). Although EF has been touted as the standard treatment of high-energy pediatric tibia fractures, our close analysis revealed a high rate of problems such as long union times (especially ages >or=12), malunion, leg length discrepancy (especially ages

Loss of renal function and microvascular blood flow after suprarenal aortic clamping and reperfusion (SPACR) above the superior mesenteric artery is greatly augmented compared with SPACR above the renal arteries.

OBJECTIVE: Renal insufficiency continues to be a complication that can affect patients after treatment for suprarenal aneurysms and renal artery occlusive disease. To our knowledge, no data are available showing that suprarenal aortic clamping and reperfusion (SRACR) above the renal arteries (renal-SRACR) preserves renal function compared with SRACR above the superior mesenteric artery (SMA-SRACR). This study examined the hypothesis that SMA-SRACR-induced downregulation of renal blood flow and function is more severe than renal-SRACR owing to the addition of systemic oxygen-derived free radical (ODFR) release. METHODS: Male Sprague-Dawley rats (about 350 g) were anesthetized and microdialysis probes or laser Doppler fibers were inserted into the renal cortex (depth of 2 mm) and into the renal medulla (depth of 4 mm). Laser Doppler blood flow was continuously monitored, and the microdialysis probes were connected to a syringe pump and perfused in vivo at 3 microL/min with lactated Ringer's solution. RESULTS: SMA-SRACR and Renal-SRACR decreased medullary and cortical blood flow and nitric oxide (NO) synthesis. SMA-SRACR downregulated cortical inducible NO synthase, whereas renal-SRACR did not. The cortex and medulla responded to the decreased blood flow and NO synthesis by increasing in prostaglandin E2 synthesis, which was due to increased cyclooxygenase-2 content. Superoxide dismutase restored SMA-SRACR (but not renal-SRACR) cortical and medullary NO synthesis, suggesting that ODFRs generated during mesenteric ischemia-reperfusion were one of the systemic mechanisms contributing to decreased renal NO synthesis in the SMA-SRACR model. The 90% decrease in creatinine clearance after SMA-SRACR was greater than the 60% decrease after renal-SRACR. CONCLUSIONS: These data show that NO is important in maintaining renal cortical and medullary blood flow and NO synthesis after renal and SMA-SRACR. These data also suggest that in addition to the renal ischemia-reperfusion caused by both models, SMA SRACR induces mesenteric ischemia-reperfusion, resulting in the generation of ODFRs, which contribute to decreased renal cortical and medullary NO synthesis. Maintaining splanchnic blood flow or attempting to keep SRACR below the SMA level may be helpful in developing strategies to minimize the renal injury after SRACR.