Substrate-induced conformational change in a trimeric ornithine transcarbamoylase

The crystal structure of Escherichia coli ornithine transcarbamoylase (OTCase, EC 2.1.3.3) complexed with the bisubstrate analog N-(phosphonacetyl)-L-ornithine (PALO) has been determined at 2.8-A resolution. This research on the structure of a transcarbamoylase catalytic trimer with a substrate analog bound provides new insights into the linkages between substrate binding, protein-protein interactions, and conformational change. The structure was solved by molecular replacement with the Pseudomonas aeruginosa catabolic OTCase catalytic trimer (Villeret, V., Tricot, C., Stalon, V. & Dideberg, O. (1995) Proc. Natl. Acad. Sci. USA 92, 10762-10766; Protein Data Bank reference pdb 1otc) as the model and refined to a crystallographic R value of 21.3%. Each polypeptide chain folds into two domains, a carbamoyl phosphate binding domain and an L-ornithine binding domain. The bound inhibitor interacts with the side chains and/or backbone atoms of Lys-53, Ser-55, Thr-56, Arg-57, Thr-58, Arg-106, His-133, Asn-167, Asp-231, Met-236, Leu-274, Arg-319 as well as Gln-82 and Lys-86 from an adjacent chain. Comparison with the unligated P. aeruginosa catabolic OTCase structure indicates that binding of the substrate analog results in closure of the two domains of each chain. As in E. coli aspartate transcarbamoylase, the 240s loop undergoes the largest conformational change upon substrate binding. The clinical implications for human OTCase deficiency are discussed.     

Circular ruffle formation and closure lead to macropinocytosis in hepatocyte growth factor/scatter factor-treated cells

Treatment with hepatocyte growth factor/scatter factor (HGF/SF) rapidly induced the formation of conspicuous circular ruffles on the apical surfaces of two kidney cell lines, MDCK and PtK2. The ruffles were found to contain significant amounts of F-actin and myosin as judged by immunofluorescence microscopy. Time-lapse photomicroscopy demonstrated that the ruffles constrict, closing over, and were followed by the formation of phase bright structures. That these structures were macropinocytotic vesicles was confirmed using fluorescein isothiocyanate (FITC)-dextran as a marker for fluid uptake. It is hypothesized that the constriction of the ruffles followed by membrane fusion causes the vesicles to form. Treatment with suramin blocked both circular ruffle formation and scattering, suggesting that ligand binding was the causal agent for ruffle formation. The drugs amiloride and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) also completely inhibited ruffle formation, suggesting that ion transport was an early consequence of HGF/SF binding and that these transport effects had a major role in the cytoskeletal changes leading to circular ruffle formation. The appearance of macropinocytotic vesicles was also blocked by amiloride treatment. Surprisingly though, subsequent scattering was not blocked by amiloride treatment, although suramin and SITS both entirely inhibited scattering.     

Geographic distribution and clinical description of leishmaniasis cases in Peru

Asymmetric acetylcholinesterase (AChE) is anchored to the basal lamina (BL) of cholinergic synapses via its collagenic tail, yet the complement of matrix receptors involved in its attachment remains unknown. The development of a novel overlay technique has allowed us to identify two Torpedo BL components that bind asymmetric AChE: a polypeptide of approximately 140 kDa and a doublet of 195-215 kDa. These were found to stain metachromatically with Coomassie blue R-250, were solubilized by acetic acid, and were sensitive to collagenase treatment. Upon sequence analysis, the 140 kDa polypeptide yielded a characteristic collagenous motif. Another AChE-binding BL constituent, identified by overlay, corresponded to a heparan sulfate proteoglycan. Lastly, we established that this proteoglycan, but not the collagenous proteins, interacted with at least one heparin binding domain of the collagenic tail of AChE. Our results indicate that at least two BL receptors are likely to exist for asymmetric AChE in Torpedo electric organ.     

Monitoring of lineage-specific chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia

Thrombogenesis in the left atrial appendage (LAA) has been related to the special morphology of this cavity and to its size and degree of dysfunction. However, no study has focused on LAA function in conjunction with left atrial (LA) function in both sinus rhythm (SR) and nonrheumatic idiopathic atrial fibrillation (AF) in relation to clinical status (cardioembolic stroke). Forty-three patients in SR (14 patients with stroke, 29 control subjects) and 45 patients in AF (27 patients with stroke, 18 control subjects) were examined by transthoracic and transesophageal echocardiography. Baseline clinical characteristics and standard transthoracic and transesophageal measurements of the LA and LAA (size, fractional area change, flow measurements, spontaneous echo contrast, and thrombus) were recorded and compared in relation to cardiac rhythm. Patients in the stroke-SR group showed a significant decrease of fractional area change in the LA (32%+/-15%) and LAA (34%+/-15%) in relation to control subjects (43%+/-10%, p = 0.035, 49%+/-13%, p = 0.006, respectively). Patients in the stroke-AF group showed significant reduction of appendage flow measurements (outward velocity = 22+/-13 vs 33+/-19 cm/sec, p = 0.036), whereas no differences were detected in the center of the LA. In multiple regression analysis, the presence of cardioembolic stroke was positively associated with the presence of spontaneous echo contrast (p = 0.0253) and spontaneous echo contrast negatively associated with appendage inward flow velocity (p<0.001). Cardioembolic stroke in patients in SR is associated with a global decrease of shortening in both cavities and in patients with AF, with a reduction of LAA flow parameters. Patients with spontaneous echo contrast, thrombus, or both showed further reduction of shortening and flow velocities in both cavities, indicating a more advanced stage of dysfunction.     

Fault tolerance in cellular automata at high fault rates

A commonly used model for fault-tolerant computation is that of cellular automata. The essential difficulty of fault-tolerant computation is present in the special case of simply remembering a bit in the presence of faults, and that is the case we treat in this paper. We are concerned with the degree (the number of neighboring cells on which the state transition function depends) needed to achieve fault tolerance when the fault rate is high (nearly 1/2). We consider both the traditional transient fault model (where faults occur independently in time and space) and a recently introduced combined fault model which also includes manufacturing faults (which occur independently in space, but which affect cells for all time). We also consider both a purely probabilistic fault model (in which the states of cells are perturbed at exactly the fault rate) and an adversarial model (in which the occurrence of a fault gives control of the state to an omniscient adversary). We show that there are cellular automata that can tolerate a fault rate 1/2−ξ (with ξ>0) with degree O((1/ξ²)log(1/ξ)), even with adversarial combined faults. The simplest such automata are based on infinite regular trees, but our results also apply to other structures (such as hyperbolic tessellations) that contain infinite regular trees. We also obtain a lower bound of Ω(1/ξ²), even with only purely probabilistic transient faults.     

3D Knitting for Pneumatic Soft Robotics

Soft robots adapt passively to complex environments due to their inherent compliance, allowing them to interact safely with fragile or irregular objects and traverse uneven terrain. The vast tunability and ubiquity of textiles has enabled new soft robotic capabilities, especially in the field of wearable robots, but existing textile processing techniques (e.g., cut-and-sew, thermal bonding) are limited in terms of rapid, additive, accessible, and waste-free manufacturing. While 3D knitting has the potential to address these limitations, an incomplete understanding of the impact of structure and material on knit-scale mechanical properties and macro-scale device performance has precluded the widespread adoption of knitted robots. In this work, the roles of knit structure and yarn material properties on textile mechanics spanning three regimes–unfolding, geometric rearrangement, and yarn stretching–are elucidated and shown to be tailorable across unique knit architectures and yarn materials. Based on this understanding, 3D knit soft actuators for extension, contraction, and bending are constructed. Combining these actuation primitives enables the monolithic fabrication of entire soft grippers and robots in a single-step additive manufacturing procedure suitable for a variety of applications. This approach represents a first step in seamlessly “printing” conformal, low-cost, customizable textile-based soft robots on-demand.     

Diagnosing organizational decision making through responsibility charting

Decision making within many organizations is becoming increasingly complex and difficult to manage. This article describes a technique called "Responsibility Charting" which can be used as a basis for understanding and improving decision making, and for enhancing the quality of communication within an organization. The authors present a case study to illustrate the types of analyses possible with this technique.     

Hepatic patatin-like phospholipase domain-containing protein 3 sequence,single nucleotide polymorphism presence,protein confirmation,and responsiveness to energy balance in dairy cows

Patatin-like phospholipase domain-containing protein 3 (PNPLA3), commonly known as adiponutrin, is part of a novel subfamily of triglyceride lipase enzymes with potential effects on triglyceride metabolism in adipose and hepatic tissues. The predicted bovine PNPLA3 sequence has been identified, but expression of the gene had not been examined. The objectives of this study were to confirm the predicted bovine PNPLA3 gene sequence, determine expression of the bovine PNPLA3 gene in response to whole-animal energy balance, identify single nucleotide polymorphisms present in dairy cows, and verify the presence of the protein in the liver. Using liver biopsy samples collected from cows at +28 d relative to calving (DRTC), RNA was isolated and used to generate a cDNA template for amplification of the entire predicted coding sequence of PNPLA3 via PCR. To determine if energy balance alters the expression of PNPLA3, RNA was isolated and mRNA expression quantified in liver samples from mid-lactation cows after a 5-d ad libitum period (n = 5) and after a subsequent 5-d 50% feed restriction period (n = 5), and in samples collected from cows at −14, +1, +14, and +28 DRTC (n = 16). The presence of PNPLA3 protein was detected by Western blot in liver protein samples collected at +28 DRTC. Expression of hepatic PNPLA3 was decreased after a period of feed restriction (8.14 vs. 1.08 ± 2.17 arbitrary units, ad libitum vs. fasted). Expression of PNPLA3 mRNA was decreased at +1 and +14 DRTC compared with −14 DRTC (23.35, 7.28, 10.17, and 14.5 ± 4.9 arbitrary units, −14, +1, +14, and +28 DRTC, respectively). The presence of PNPLA3 protein was detected as a 55-kDa band in hepatic protein isolations from liver tissue collected at +28 DRTC. These data confirm the presence and sequence of the bovine hepatic PNPLA3 gene and single nucleotide polymorphisms. Furthermore, these data indicate responsiveness of bovine hepatic PNPLA3 to energy balance.