Design of Efficient Artificial Enzymes Using Crystallographically Enhanced Conformational Sampling.

The ability to create efficient artificial enzymes for any chemical reaction is of great interest. Here, we describe a computational design method for increasing the catalytic efficiency of de novo enzymes by several orders of magnitude without relying on directed evolution and high-throughput screening. Using structural ensembles generated from dynamics-based refinement against X-ray diffraction data collected from crystals of Kemp eliminases HG3 (kcat/KM 125 M-1 s-1) and KE70 (kcat/KM 57 M-1 s-1), we design from each enzyme ≤10 sequences predicted to catalyze this reaction more efficiently. The most active designs display kcat/KM values improved by 100-250-fold, comparable to mutants obtained after screening thousands of variants in multiple rounds of directed evolution. Crystal structures show excellent agreement with computational models, with catalytic contacts present as designed and transition-state root-mean-square deviations of ≤0.65 Å. Our work shows how ensemble-based design can generate efficient artificial enzymes by exploiting the true conformational ensemble to design improved active sites.

Design of efficient artificial enzymes using crystallographically-enhanced conformational sampling.

The ability to create efficient artificial enzymes for any chemical reaction is of great interest. Here, we describe a computational design method for increasing catalytic efficiency of de novo enzymes to a level comparable to their natural counterparts without relying on directed evolution. Using structural ensembles generated from dynamics-based refinement against X-ray diffraction data collected from crystals of Kemp eliminases HG3 (kcat/KM 125 M-1 s-1) and KE70 (kcat/KM 57 M-1 s-1), we design from each enzyme ≤10 sequences predicted to catalyze this reaction more efficiently. The most active designs display kcat/KM values improved by 100-250-fold, comparable to mutants obtained after screening thousands of variants in multiple rounds of directed evolution. Crystal structures show excellent agreement with computational models. Our work shows how computational design can generate efficient artificial enzymes by exploiting the true conformational ensemble to more effectively stabilize the transition state.

Computational remodeling of an enzyme conformational landscape for altered substrate selectivity.

Structural plasticity of enzymes dictates their function. Yet, our ability to rationally remodel enzyme conformational landscapes to tailor catalytic properties remains limited. Here, we report a computational procedure for tuning conformational landscapes that is based on multistate design of hinge-mediated domain motions. Using this method, we redesign the conformational landscape of a natural aminotransferase to preferentially stabilize a less populated but reactive conformation and thereby increase catalytic efficiency with a non-native substrate, resulting in altered substrate selectivity. Steady-state kinetics of designed variants reveals activity increases with the non-native substrate of approximately 100-fold and selectivity switches of up to 1900-fold. Structural analyses by room-temperature X-ray crystallography and multitemperature nuclear magnetic resonance spectroscopy confirm that conformational equilibria favor the target conformation. Our computational approach opens the door to targeted alterations of conformational states and equilibria, which should facilitate the design of biocatalysts with customized activity and selectivity.

Performance of the adaptive optics system for Laser Communications Relay Demonstration's Ground Station 1.

The Laser Communications Relay Demonstration is NASA's multi-year demonstration of laser communication from the Earth to a geosynchronous satellite. The mission currently has two optical ground stations (OGSs), with one in California (OGS1) and one in Hawaii (OGS2). Each ground terminal optical system consists of a high-order adaptive optics (AO) system, a laser transmit system, and a camera for target acquisition. The OGS1 AO system is responsible for compensating for the downlink beam for atmospheric turbulence and coupling it into the modem's single mode fiber. The mission requires a coupling efficiency of 50%, which necessitates a high-order AO system. To achieve this performance, the AO system uses two deformable mirrors with one mirror correcting for low-spatial-frequency aberrations with large amplitude and a second deformable mirror correcting for high-spatial-frequency aberrations with small amplitude. Turbulence is sensed with a Shack-Hartmann wavefront sensor. To meet its performance requirements in the most stressing conditions, the system can operate at frame rates of 20 kHz. This high frame rate is enabled by the design of the real-time control system. We present an overview of both the hardware and software design of the system, and describe the control system and methods of reducing non-common path aberrations. Finally, we show measured system performance.

All-MEMS Lidar Using Hybrid Optical Architecture with Digital Micromirror Devices and a 2D-MEMS Mirror.

In a lidar system, replacing moving components with solid-state devices is highly anticipated to make a reliable and compact lidar system, provided that a substantially large beam area with a large angular extent as well as high angular resolution is assured for the lidar transmitter and receiver. A new quasi-solid-state lidar optical architecture employs a transmitter with a two-dimensional MEMS mirror for fine beam steering at a fraction of the degree of the angular resolution and is combined with a digital micromirror device for wide FOV scanning over 37 degree while sustaining a large aperture area of 140 mm squared. In the receiver, a second digital micromirror device is synchronized to the transmitter DMD, which enables a large FOV receiver. An angular resolution of 0.57°(H) by 0.23° (V) was achieved with 0.588 fps for scanning 1344 points within the field of view.

Single-chip holographic beam steering for lidar by a digital micromirror device with angular and spatial hybrid multiplexing.

A digital micromirror device (DMD) based holographic beam steering technique is reported that multiplexes fine-steering binary amplitude gratings with a coarse-steering programmable blazed grating. The angular spatial light modulation (ASLM) technique encodes the spatial pattern of the binary amplitude grating at the same plane as the angular modulation set by a phase map of the DMD-based beam steering technique. The beam steering technique is demonstrated at 532 nm and implemented into a 905 nm lidar system. The results of the lidar system tests are presented, achieving a 44° field-of-view, 0.9°×0.4° (H×V) angular resolution, 1 m max distance, 1.5 kHz sampling, and 7.8 FPS video. Scalability techniques are proposed, including max distance increases to over 100 m.

Fast laser beam steering into multiple diffraction orders with a single digital micromirror device for time-of-flight lidar.

The sampling rate and angular resolution of diffraction-based beam steering employing a digital micromirror device (DMD) can be simultaneously enhanced by at least an order of magnitude by synchronizing multiple nanosecond laser sources and pulses during each DMD actuation. A time-of-flight single-chip DMD lidar with three sources measures a range at a 3.34 kHz sampling rate and a 3.4° angular resolution across a 48° field of view. Employing multiple diffraction orders of the DMD improves the sampling rate at least by a factor of 2 and up to the number of diffraction orders supported by the DMD. An improved sampling rate of 6.68 kHz with a 9.6° angular resolution is experimentally demonstrated by illuminating micromirrors multiple times within a single transition period of the micromirrors.

Retrospective Analysis of HER2+ Breast Cancer Outcomes at a County Hospital: Do Published Outcomes Hold up in the Real World?

INTRODUCTION: Landmark trials repeatedly demonstrate that pertuzumab and trastuzumab plus standard chemotherapy have the best outcomes in human epidermal growth factor receptor 2 (HER2) positive breast cancer in the neoadjuvant, adjuvant, and metastatic setting. However, many of these multicenter landmark trials lack diversity and studied largely Caucasian populations. Our goal is to address this under-representation of minorities, and compare pathologic complete response (pCR) rates in our predominantly Hispanic population with HER2 positive breast cancer receiving the same neoadjuvant chemotherapy (NACT) at Olive View-UCLA Medical Center (OVMC) to that of pCR rates observed in the TRYPHAENA trial. METHODS: For this retrospective cohort study, we compiled a list of 53 patients aged 18 and older, 52 female and 1 male, with HER2 positive breast cancer identified by fluorescence in situ hybridization treated at OVMC from December 2015 to May 2018. Our population was 57% Hispanic, 13% white, 13% Filipino, 11% Asian, 2% black, and 4% other. The complete list included patients receiving standard neoadjuvant, adjuvant, and metastatic chemotherapy regimens. We analyzed 23 female patients with HER2 positive breast cancer staged I to IIIC, receiving standard NACT (docetaxel, carboplatin, trastuzumab, and pertuzumab). Metastatic HER2 positive breast cancer patients were excluded. The primary outcome studied was pCR rates after receiving NACT. pCR was defined as the absence of invasive cancer cells from tissue samples removed after surgery. Secondary outcomes measured were side effects of chemotherapy. pCR rates and side effects were compared to TRYPHAENA. Data regarding insurance status, breast cancer detection modality, and time to seek medical attention were recorded. RESULTS: 50% of our patients who received NACT achieved pCR. Our pCR rates mirrored those observed in the TRYPHAENA trial (51.9%). The most common side effect observed in our population was diarrhea. A higher proportion (37.5%) of our patients had liver function test (LFT) elevation compared to the TRYPHAENA trial (3.9%). Baseline LFTs were normal prior to treatment in 96% of patients. In terms of modality of detection, 70% were self-palpated, 26% were detected through routine mammography, and 4% were found incidentally. Average time from mass discovery to seeking medical attention was 3.4 months. Only 26% had medical insurance at diagnosis. Although not included in our study, 28% of our patients were initially diagnosed with stage IV metastatic disease.  Conclusion: Our study found that pCR rates in our primarily Hispanic population compared well to the response rates observed in landmark trials with largely Caucasian populations. Genetic variations in chemo-sensitivity may have a minimal influence on cancer care outcomes in this population.

Prostate Cancer Metastatic to the Peritoneum: A Road Less Traveled by a Common Malignancy.

A 70-year-old Indian male with a history of a Gleason 7 (3+4) prostate cancer presented with abdominal ascites. Imaging was remarkable for peritoneal carcinomatosis as well as possible metastases to the bladder and seminal vesicle. Given the atypical pattern of presentation, further investigation was performed with studies of the ascites fluid. Cytology from the ascites fluid returned consistent with malignant cells of prostatic origin. His treatment course included androgen deprivation therapy (ADT), docetaxel, abiraterone, and cabazitaxel. He had eventual progression and worsening of his disease and performance status and was transitioned to hospice. This case demonstrated the importance of pursuing a thorough diagnostic evaluation, when faced with a rare presentation of a common malignancy. Furthermore, it illustrated the challenges incurred when tailoring standard regimens to best address the needs of the whole patient and not simply their disease.

Brachytherapy in pelvic malignancies: a review for radiologists.

Brachytherapy, also known as sealed source or internal radiation therapy, involves placement of a radioactive source immediately adjacent to or within tumor, thus enabling delivery of a localized high dose of radiation. Compared with external beam radiation which must first pass through non-target tissues, brachytherapy results in less radiation dose to normal tissues. In the past decade, brachytherapy use has markedly increased, thus radiologists are encountering brachytherapy devices and their associated post-treatment changes to increasing degree. This review will present a variety of brachytherapy devices that radiologists may encounter during diagnostic pelvic imaging with a focus on prostate and gynecologic malignancies. The reader will become familiar with the function, correct position, and potential complications of brachytherapy devices in an effort to improve diagnostic reporting and communication with clinicians.

Engineering alginate as bioink for bioprinting.

Recent advances in three-dimensional (3-D) printing offer an excellent opportunity to address critical challenges faced by current tissue engineering approaches. Alginate hydrogels have been used extensively as bioinks for 3-D bioprinting. However, most previous research has focused on native alginates with limited degradation. The application of oxidized alginates with controlled degradation in bioprinting has not been explored. Here, a collection of 30 different alginate hydrogels with varied oxidation percentages and concentrations was prepared to develop a bioink platform that can be applied to a multitude of tissue engineering applications. The authors systematically investigated the effects of two key material properties (i.e. viscosity and density) of alginate solutions on their printabilities to identify a suitable range of material properties of alginates to be applied to bioprinting. Further, four alginate solutions with varied biodegradability were printed with human adipose-derived stem cells (hADSCs) into lattice-structured, cell-laden hydrogels with high accuracy. Notably, these alginate-based bioinks were shown to be capable of modulating proliferation and spreading of hADSCs without affecting the structure integrity of the lattice structures (except the highly degradable one) after 8days in culture. This research lays a foundation for the development of alginate-based bioink for tissue-specific tissue engineering applications.

Rapid discontinuation of corticosteroids in pediatric renal transplantation.

Corticosteroid immunosuppression has permitted the development of successful allotransplantation; however, corticosteroids are associated significant post-transplant complications. To circumvent these problems, we implemented a protocol of rapid discontinuation of corticosteroids in 19 consecutive pediatric primary kidney transplant recipients. Mean age at time of transplant was 13.4 (+/-4.5) yr, 52.6% were male, 63.2% underwent living donor transplantation. All patients were administered Thymoglobulin [anti-thymocyte globulin (rabbit)] as induction immunosuppression with a rapid tapering dose of corticosteroids (total of five daily doses), and maintained on mycophenolate mofetil and tacrolimus. Two patients had immediate recurrence of primary disease (FSGS), requiring further corticosteroid therapy. Otherwise, remaining 17 patients were maintained off corticosteroids, with excellent graft function; mean baseline eGFR of 112 mL/min/1.73 m(2) (+/-19) at 28 months (+/-14) post-transplantation. There was 100% patient and rejection-free graft survival at 27 months (range 5-58 months) post-transplantation; 47% underwent renal transplant biopsy secondary to acute rise in serum creatinine with or without worsening hypertension. All biopsies had no evidence of acute rejection; 62.5% had findings consistent with tacrolimus toxicity. Renal transplantation utilizing a rapid discontinuation of corticosteroid protocol in pediatric patients appears to be safe and effective, without increasing the risk of acute rejection or graft loss.

The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3-E1 osteoblasts.

Fluid flow due to loading in bone is a potent mechanical signal that may play an important role in bone adaptation to its mechanical environment. Previous in vitro studies of osteoblastic cells revealed that the upregulation of cyclooxygenase-2 (COX-2) and c-fos induced by steady fluid flow depends on a change in actin polymerization dynamics and the formation of actin stress fibers. Exposing cells to dynamic oscillatory fluid flow, the temporal flow pattern that results from normal physical activity, is also known to result in increased COX-2 expression and PGE(2) release. The purpose of this study was to determine whether dynamic fluid flow results in changes in actin dynamics similar to steady flow and to determine whether alterations in actin dynamics are required for PGE(2) release. We found that exposure to oscillatory fluid flow did not result in the development of F-actin stress fibers in MC3T3-E1 osteoblastic cells and that inhibition of actin polymerization with cytochalasin D did not inhibit intracellular calcium mobilization or PGE(2) release. In fact, PGE(2) release was increased threefold in the polymerization inhibited cells and this PGE(2) release was dependent on calcium release from the endoplasmic reticulum. This was in contrast to the PGE(2) release that occurs in normal cells, which is independent of calcium flux from endoplasmic reticulum stores. We suggest that this increased PGE(2) release involves a different molecular mechanism perhaps involving increased deformation due to the compromised cytoskeleton.