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This study demonstrated successful encapsulation of polymerized hemoglobin in the tense quaternary state inside a nanoparticle comprised of zeolite imidazole framework precursors with antioxidant properties.

Monocular 3D human pose estimation is challenging due to depth ambiguity. Convolution-based and Graph-Convolution-based methods have been developed to extract 3D information from temporal cues in motion videos. Typically, in the lifting-based methods, most recent works adopt the transformer to model the temporal relationship of 2D keypoint sequences. These previous works usually consider all the joints of a skeleton as a whole and then calculate the temporal attention based on the overall characteristics of the skeleton. Nevertheless, the human skeleton exhibits obvious part-wise inconsistency of motion patterns. It is therefore more appropriate to consider each part's temporal behaviors separately. To deal with such part-wise motion inconsistency, we propose the Part Aware Temporal Attention module to extract the temporal dependency of each part separately. Moreover, the conventional attention mechanism in 3D pose estimation usually calculates attention within a short time interval. This indicates that only the correlation within the temporal context is considered. Whereas, we find that the part-wise structure of the human skeleton is repeating across different periods, actions, and even subjects. Therefore, the part-wise correlation at a distance can be utilized to further boost 3D pose estimation. We thus propose the Part Aware Dictionary Attention module to calculate the attention for the part-wise features of input in a dictionary, which contains multiple 3D skeletons sampled from the training set. Extensive experimental results show that our proposed part aware attention mechanism helps a transformer-based model to achieve state-of-the-art 3D pose estimation performance on two widely used public datasets. The codes and the trained models are released at https://github.com/thuxyz19/3D-HPE-PAA.

Various types of hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as red blood cell substitutes for treating blood loss when blood is not available. Among those HBOCs, glutaraldehyde polymerized Hbs have attracted significant attention due to their facile synthetic route, and ability to expand the blood volume and deliver oxygen. Hemopure®, Oxyglobin®, and PolyHeme® are the most well-known commercially developed glutaraldehyde polymerized Hbs. Unfortunately, only Oxyglobin® was approved by the FDA for veterinary use in the United States, while Hemopure® and PolyHeme® failed phase III clinical trials due to their ability to extravasate from the blood volume into the tissue space which facilitated nitric oxide scavenging and tissue deposition of iron, which elicited vasoconstriction, hypertension and oxidative tissue injury. Fortunately, conjugation of poly (ethylene glycol) (PEG) on the surface of Hb is capable of reducing the vasoactivity of Hb by creating a hydration layer surrounding the Hb molecule, which increases its hydrodynamic diameter and reduces tissue extravasation. Several commercial PEGylated Hbs (MP4®, Sanguinate®, Euro-PEG-Hb) have been developed for clinical use with a longer circulatory half-life and improved safety compared to Hb. However, all of these commercial products exhibited relatively high oxygen affinity compared to Hb, which limited their clinical use. To dually address the limitations of prior generations of polymerized and PEGylated Hbs, this current study describes the PEGylation of polymerized bovine Hb (PEG-PolybHb) in both the tense (T) and relaxed (R) quaternary state via thiol-maleimide chemistry to produce an HBOC with low or high oxygen affinity. The biophysical properties of PEG-PolybHb were measured and compared with those of commercial polymerized and PEGylated HBOCs. T-state PEG-PolybHb possessed higher hydrodynamic volume and P50 than previous generations of commercial PEGylated Hbs. Both T- and R-state PEG-PolybHb exhibited significantly lower haptoglobin binding rates than the precursor PolybHb, indicating potentially reduced clearance by CD163 + monocytes and macrophages. Thus, T-state PEG-PolybHb is expected to function as a promising HBOC due to its low oxygen affinity and enhanced stealth properties afforded by the PEG hydration shell.

Polymerized human hemoglobin (PolyhHb) is being studied as a possible red blood cell (RBC) substitute for use in scenarios where blood is not available. While the oxygen (Osub2/sub) carrying capacity of PolyhHb makes it appealing as an Osub2/subtherapeutic, the commercial PolyhHb PolyHeme® (Northfield Laboratories Inc.) was never approved for clinical use due to the presence of large quantities of low molecular weight (LMW) polymeric hemoglobin (Hb) species (lt;500 kDa), which have been shown to elicit vasoconstriction, systemic hypertension, and oxidative tissue injury in vivo. Previous bench-top scale studies in our lab demonstrated the ability to synthesize and purify PolyhHb using a two-stage tangential flow filtration purification process to remove almost all undesirable Hb species (gt;0.2 µm andlt;500 kDa) in the material, to create a product that should be safer for transfusion. Therefore, to enable future large animal studies and eventual human clinical trials, PolyhHb synthesis and purification processes need to be scaled up to the pilot scale. Hence in this study, we describe the pilot scale synthesis and purification of PolyhHb. Characterization of pilot scale PolyhHb showed that PolyhHb could be successfully produced to yield biophysical properties conducive for its use as an RBC substitute. Size exclusion high performance liquid chromatography showed that pilot scale PolyhHb yielded a high molecular weight Hb polymer containing a small percentage of LMW Hb species (lt;500 kDa). Additionally, the auto-oxidation rate of pilot scale PolyhHb was even lower than that of previous generations of PolyhHb. Taken together, these results demonstrate that PolyhHb has the ability to be seamlessly manufactured at the pilot scale to enable future large animal studies and clinical trials.

Previously, our lab developed high molecular weight (MW) tense (T) quaternary state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this work, we prepared PolybHb in the T- and relaxed (R) quaternary state with ultrahigh MW (> 500 kDa) with varying cross-link densities and investigated the effect of MW on key biophysical properties (i.e., O(2) affinity, cooperativity (Hill) coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand-binding kinetics, auto-oxidation, and haptoglobin-binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta-data analysis to evaluate correlations between procedural parameters (i.e. cross-linker:bovine Hb (bHb) molar ratio, gas/liquid exchange time, temperature during sodium dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T-state and R-state PolybHbs. Our results showed that, the duration of the fast-step auto-oxidation phase of R-state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHbs exhibited significantly higher bimolecular rate constants for binding to haptoglobin and unimoleular O(2) offloading rate constants compared to R-state PolybHbs. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHbs. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.

Polymerized human hemoglobin (PolyhHb) is being studied as a possible red blood cell (RBC) substitute for use in scenarios where blood is not available. While the O carrying capacity of PolyhHb makes it appealing as an O therapeutic, the commercial PolyhHb PolyHeme® (Northfield Laboratories Inc., Evanston, IL) was never approved for clinical use due to the presence of large quantities of low molecular weight polymeric (LMW) Hb species (in vivo. Previous bench-top scale studies in our lab demonstrated the ability to synthesize and purify PolyhHb using a two-stage tangential flow filtration (TFF) purification process to remove almost all undesirable Hb species (>0.2 µm and

Methemoglobin (metHb) arises from the oxidation of ferrous hemoglobin (HbFe2+, Hb) to ferric hemoglobin (HbFe3+, metHb), which is unable to bind gaseous ligands such as oxygen (O2) and carbon monoxide (CO), and binds to nitric oxide (NO) significantly slower compared to Hb. Therefore, metHb does not elicit vasoconstriction and systemic hypertension in vivo due to its extremely slow NO scavenging rate in comparison to cell-free Hb, but will induce oxidative tissue injury, demonstrating the potential of using metHb as a control material when studying the toxicity of cell-free Hb. Hence, the goal of this work was to develop a novel manufacturing strategy for production of metHb that is amenable to scale-up. In this study, small scale (e.g. 1 mL reaction volume) screening experiments were initially conducted to determine the optimal molar ratio of Hb to the oxidization agents hydrogen peroxide (H2O2) or sodium nitrite (NaNO2) to achieve the highest conversion of Hb into metHb. A spectral deconvolution program was employed to determine the molar fraction of various species (hemichrome, metHb, oxyHb, metHb-NO2−, and NaNO2) in solution during the oxidation reaction. From this analysis, either a 1:1 or 1:5 molar ratio was identified as optimal molar ratios of Hb:NaNO2 (heme basis) that yielded the highest conversion of Hb into metHb with negligible amounts of side products. Hence in order to reduce the reaction time, a 1:5 molar ratio was chosen for large scale (i.e. 1.5 L reaction volume) synthesis of bovine metHb (metbHb) and human metHb (methHb). The biophysical properties of metHb were then characterized to elucidate the potential of using the synthesized metHb as a non-O2 carrying control material. The haptoglobin binding kinetics of metHb were found to be similar to Hb. Additionally, the synthesized metHb was stable in phosphate buffered saline (PBS, 50 mM, pH 7.4) at 4°C for approximately one week, indicating the high stability of the material.

BACKGROUND: Tissue ischemia contributes to necrosis and infection. While angiogenic cell therapies have emerged as a promising strategy against ischemia, current approaches to cell therapies face multiple hurdles. Recent advances in nuclear reprogramming could potentially overcome some of these limitations. However, under severely ischemic conditions necrosis could outpace reprogramming-based repair. As such, adjunctive measures are required to maintain a minimum level of tissue viability/activity for optimal response to restorative interventions. METHODS: Here we explored the combined use of polymerized hemoglobin (PolyHb)-based oxygen nanocarriers with Tissue Nano-Transfection (TNT)-driven restoration to develop tissue preservation/repair strategies that could potentially be used as a first line of care. Random-pattern cutaneous flaps were created in a mouse model of ischemic injury. PolyHbs with high and low oxygen affinity were synthesized and injected into the tissue flap at various timepoints of ischemic injury. The degree of tissue preservation was evaluated in terms of perfusion, oxygenation, and resulting necrosis. TNT was then used to deploy reprogramming-based vasculogenic cell therapies to the flaps via nanochannels. Reprogramming/repair outcomes were evaluated in terms of vascularity and necrosis. RESULTS: Flaps treated with PolyHbs exhibited a gradual decrease in necrosis as a function of time-to-intervention, with low oxygen affinity PolyHb showing the best outcomes. TNT-based intervention of the flap in combination with PolyHb successfully curtailed advanced necrosis compared to flaps treated with only PolyHb or TNT alone. CONCLUSIONS: These results indicate that PolyHb and TNT technologies could potentially be synergistically deployed and used as early intervention measures to combat severe tissue ischemia. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-020-00621-4) contains supplementary material, which is available to authorized users.

The photon beam lines from beam port 3B1 recently constructed at Beijing Synchrotron Radiation Laboratory (BSRL) are beam lines for soft x‐ray lithography and VUV‐visible spectroscopy alternately. The commissioning of these beam lines has been carried out under both the parasitic and dedicated modes of the storage ring Beijing electron‐positron collider (BEPC). This article reports the status of these two beam lines as well as the comparison of the preliminary measurement results with the calculated synchrotron radiation spectra of these beam lines.

At present beamline 3B1 in Synchrotron Radiation Lab. of Beijing Electron Position Collider has been set up for soft X-ray photolithography experiment. 3B1B beamline branched from beamline 3B1 operates at VUV-VIS for biological spectroscopic experiment. This paper describes design and fabrication on optical system of 3B1B. The features of optical system design is to adopt an off-axis paraboloid mirror which focuses light beam from synchrotron radiation source on entrance slit of Vm-562 vacuum monochromator and homogeneously circular spot appears on focal plane. An ellipsoidal mirror is used to focus spot on exit slit onto sample. It is very difficult to fabrication two aspherical mirror, it is 600 mm from axis, focal length f equals 315.7, dimension 40 X 100 mm2, length of long axis of the ellipsoidal mirror is 1200 mm and that of short axis is 848.52 mm. Al+MgF2 layer is coated on the surface mirror. We make use of special fabrication technology and testing method for mirrors and get satisfied with results. Figure error of two mirrors: diameter of circle of diffusion is less than 0.1 mm and surface roughness is less than 1 nm RMS. As it operates at 140 - 400 nm, incidence angle (theta) equals 45 degree(s), reflectivity of the mirror is better than 75%. These conditions meet requirements for biological spectroscopic experiments in the beamline.© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The bacteriorhodopsin (BR) molecules in the purple membrane (PM) are organized into a two-dimensional hexagonal lattice of trimers which are surrounded by about 30 lipid molecules. The protein-protein and proteinlipid interactions may affect their structure and function. It is therefore interesting to solubilize the PM into BR monomers and to investigate its functional characteristics. The BR monomers were reconstituted with dimyristoylphosphatidylcholine (DMPC) to form vesicles. We studied the pH dependence of the photoelectric response in a bilayer lipid membrane (BLM) system and of the kinetics of the M412 and the 0640 photocycle intermediates both in PM fragments and in BR monomer vesicles. The results show that the conformation of BR in monomeric state is more readily affected by the pH of the medium. Especially in pH higher than 9.4 the M412 slow-decaying component becomes very slow and even the polarity of the photovoltage signal is reversed. It appears that BR has two different conformational forms and their distribution depends on the pH of the medium. The two forms have different photocycle intermediates with different lifetimes and even the direction of proton pumping is reversed.