In situ structure determination at nanometer resolution using TYGRESS.

The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the subtomograms. By contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We report a method called 'tomography-guided 3D reconstruction of subcellular structures' (TYGRESS) that is a hybrid of cryo-ET and SP-cryo-EM, and is able to achieve close-to-nanometer resolution of complexes inside crowded cellular environments. TYGRESS combines the advantages of SP-cryo-EM (images with good signal-to-noise ratio and contrast, as well as minimal radiation damage) and subtomogram averaging (three-dimensional alignment of macromolecules in a complex sample). Using TYGRESS, we determined the structure of the intact ciliary axoneme with up to resolution of 12 Å. These results reveal many structural details that were not visible by cryo-ET alone. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging.

A Crowdsourced Evaluation of Facial Averageness and Attractiveness.

BACKGROUND: Evolutionary psychologists have demonstrated that humans are attracted to individuals who possess average anatomy for the population. OBJECTIVES: The aim of this study was to prove that a composite of average facial features would be more attractive to raters than the cohort utilized to create the composite. METHODS: The male and female cohorts each consisted of 41 standardized frontal-view monochrome photographs, with 1 composite image derived from the other 40 real images. Amazon Mechanical Turk, a widely used crowdsourcing platform, was utilized to obtain ratings of images ranging from 1 to 7, with 1 and 7 being least and most attractive, respectively. The strength of the preference for the composite over the real images was assessed by the difference between the mean rating of the composite and real images. RESULTS: In total, 870 and 876 respondents were recruited to rate the male and female cohorts, respectively. For the male and female cohorts, the composite image was rated significantly higher than the rest of the cohort overall and across all ages, genders, and countries of residence (all P < 0.0001). For both cohorts, the strength of the preference was significantly higher for European respondents and lower for South American and nonbinary respondents (all P < 0.05). CONCLUSIONS: This study reveals that average facial anatomy is perceived as most attractive across all demographics, a finding that is hoped to serve as a stepping stone for further studies leading to objective cosmetic quantifications and integrating evidence-based medicine into aesthetic surgery.

Heterozygous Mutation of Vegfr3 Reduces Renal Lymphatics without Renal Dysfunction.

BACKGROUND: Lymphatic abnormalities are observed in several types of kidney disease, but the relationship between the renal lymphatic system and renal function is unclear. The discovery of lymphatic-specific proteins, advances in microscopy, and available genetic mouse models provide the tools to help elucidate the role of renal lymphatics in physiology and disease. METHODS: We utilized a mouse model containing a missense mutation in Vegfr3 (dubbed Chy ) that abrogates its kinase ability. Vegfr3 Chy/+ mice were examined for developmental abnormalities and kidney-specific outcomes. Control and Vegfr3 Chy/+ mice were subjected to cisplatin-mediated injury. We characterized renal lymphatics using tissue-clearing, light-sheet microscopy, and computational analyses. RESULTS: In the kidney, VEGFR3 is expressed not only in lymphatic vessels but also, in various blood capillaries. Vegfr3 Chy/+ mice had severely reduced renal lymphatics with 100% penetrance, but we found no abnormalities in BP, serum creatinine, BUN, albuminuria, and histology. There was no difference in the degree of renal injury after low-dose cisplatin (5 mg/kg), although Vegfr3 Chy/+ mice developed perivascular inflammation. Cisplatin-treated controls had no difference in total cortical lymphatic volume and length but showed increased lymphatic density due to decreased cortical volume. CONCLUSIONS: We demonstrate that VEGFR3 is required for development of renal lymphatics. Our studies reveal that reduced lymphatic density does not impair renal function at baseline and induces only modest histologic changes after mild injury. We introduce a novel quantification method to evaluate renal lymphatics in 3D and demonstrate that accurate measurement of lymphatic density in CKD requires assessment of changes to cortical volume.

A general algorithm for consensus 3D cell segmentation from 2D segmented stacks.

Cell segmentation is the fundamental task. Only by segmenting, can we define the quantitative spatial unit for collecting measurements to draw biological conclusions. Deep learning has revolutionized 2D cell segmentation, enabling generalized solutions across cell types and imaging modalities. This has been driven by the ease of scaling up image acquisition, annotation and computation. However 3D cell segmentation, which requires dense annotation of 2D slices still poses significant challenges. Labelling every cell in every 2D slice is prohibitive. Moreover it is ambiguous, necessitating cross-referencing with other orthoviews. Lastly, there is limited ability to unambiguously record and visualize 1000's of annotated cells. Here we develop a theory and toolbox, u-Segment3D for 2D-to-3D segmentation, compatible with any 2D segmentation method. Given optimal 2D segmentations, u-Segment3D generates the optimal 3D segmentation without data training, as demonstrated on 11 real life datasets, >70,000 cells, spanning single cells, cell aggregates and tissue.

Pyridine-bridged cobalt tetra-aminophthalocyanine to active peroxymonosulphate for efficient degrading carbamazepine.

In this study, each cobalt tetra-aminophthalocyanine (CoTAPc) molecule was immobilised with four isonicotinic acid (INA) molecules by amide bonding, a novel and highly efficient catalyst pyridine-bridged cobalt tetra-aminophthalocyanine (CoTAPc-TINA) was synthesised. The introduction of INA molecules promoted CoTAPc to expose more active sites, and increased the electron cloud density of cobalt ions promoting O-O bond homolysis of PMS to generate more active species, which significantly enhanced catalytic activity. With the pharmaceutical of carbamazepine (CBZ) as model pollutant, 0.1 g/L CoTAPc-TINA in dark in the presence of 0.4 mM PMS, 98.8% CBZ was removed within 10 min. However, under the same conditions the removed of CBZ was only 58.9% by CoTAPc/PMS system. Radical capture experiments combined electron paramagnetic resonance technology demonstrate that hydroxyl radicals, sulphate radicals, superoxide radicals and singlet oxygen are the main active species in the CoTAPc-TINA/PMS system. As the reaction proceeded, all aromatic intermediates were transformed to small molecular acids by these active species. This investigation provided a new insight for application of metal phthalocyanine in wastewater treatment.

Facial Nerve High-Resolution Microanatomy Analysis of the Distal Intratemporal to Extracranial Pes Segment: Reconstructive Implications.

BACKGROUND: Current knowledge of facial nerve topography between the stylomastoid foramen to the pes anserinus is very limited. Elucidating this segment's intraneural microanatomy may be advantageous in certain clinical settings: the planning of nerve grafts for gaps extending from the proximal facial nerve trunk to distal branches or in determining coaptation sites for hypoglossal jump grafts to provide selective upper and lower facial tone. This study is the first to provide high-definition intraneural topography of the aforementioned segment to optimize reconstructive outcomes. METHODS: Sixteen facial nerves extending from the second genu to the pes anserinus were harvested from eight cadavers en bloc to preserve orientation. Specimens were imaged by micro-computed tomography using a serial 6-µm protocol and digitally reconstructed three-dimensionally to be analyzed using bioinformatic tools. RESULTS: No clinically significant fascicular separation was noted between 14.4 mm proximal to the stylomastoid foramen until 4.4 mm distal to the foramen. Fascicles remained separate throughout the remainder of the specimen and were found to undergo a mean rotation of 45.5 degrees ( P = 0.0002) between 8.9 and 13.7 mm distal to the stylomastoid foramen. This reliable clockwise rotation in left nerves and counterclockwise rotation in right nerves resulted in superficially oriented fascicles entering the upper division of the pes anserinus, whereas deep-oriented fascicles entered the lower division. CONCLUSION: Intraneural facial nerve topography and rotation are consistent from 4 to 14 mm distal to the stylomastoid foramen, enabling surgeons to accurately place grafts targeted to either the upper or lower face, thus optimizing functional accuracy and minimizing synkinesis.

Hydration-layer models for cryo-EM image simulation.

To compare cryo-EM images and 3D reconstructions with atomic structures in a quantitative way it is essential to model the electron scattering by solvent (water or ice) that surrounds protein assemblies. The most rigorous method for determining the density of solvating water atoms for this purpose has been to perform molecular-dynamics (MD) simulations of the protein-water system. In this paper we adapt the ideas of bulk-water modeling that are used in the refinement of X-ray crystal structures to the cryo-EM solvent-modeling problem. We present a continuum model for solvent density which matches MD-based results to within sampling errors. However, we also find that the simple binary-mask model of Jiang and Brünger (1994) performs nearly as well as the new model. We conclude that several methods are now available for rapid and accurate modeling of cryo-EM images and maps of solvated proteins.

The Impact of Comorbidities on Patient Outcomes in the Upper Body Lift: A Retrospective Review.

Background: Nonsurgical and surgical weight loss options have improved over the past several decades resulting in an increased number of patients who present with body contour deformities. This review focuses on the upper truncal deformity. This deformity is discernable by its residual tissue laxity in the upper arm, back, lateral chest, and breast. Objective: The purpose of this study is to evaluate the morbidity of this procedure when these regions are treated in one operative setting. Methods: A retrospective chart review of patients who underwent an upper body lift for truncal deformities after massive weight loss by the senior author between August 2006 and December 2019 was performed. Patient comorbidities and demographics, preoperative parameters, operative factors, and minor and major complications were assessed. Results: No intraoperative or major complications occurred. The overall complication rate was 71% (20/28), which were all minor and most related to wound breakdown. Using logistical regression analysis, we found that neither BMI nor amount of weight resected contributed to a higher complication rate in this cohort. Simple matching coefficients analysis identified anemia, hypertension, lifetime smoking history, celecoxib use, and multiple concurrent procedures as comorbidities and intraoperative factors with an increased risk for adverse outcomes. Conclusions: This review helps define the role of upper body lift in the care of patients with massive weight loss and addresses the morbidity of a comprehensive approach to upper body deformity. Appropriate patient selection, preoperative patient counseling, sound operative technique, and supportive postoperative care can help to avoid adverse outcomes.

Operative Time Predicts Postoperative Outcomes in Bilateral DIEP Flap Reconstruction: Multivariate 1000 Flap Analysis.

Skin-to-skin operative time (OT) as a risk factor for adverse postoperative outcomes in microvascular breast reconstruction has not been thoroughly investigated. This study evaluates OT's impact on length of stay (LOS), overall morbidity, individual complications, and unplanned reoperation (UR) in deep inferior epigastric artery perforator (DIEP) flaps, with a primary objective of identifying a clinically relevant time of decreased odds. Methods: Patients who underwent bilateral DIEP flaps from 2010 to 2021 by two senior surgeons (N.T.H. and S.S.T.) with standardized surgical and postoperative protocols were retrospectively reviewed. One thousand flaps (500 patients) were analyzed with extensive multivariate regression equations to adjust for potential confounders, including intraoperative complexity. The odds of postoperative complication, extended LOS (eLOS, defined as ≥5 days) were compared across OT per hour and OT intervals. Results: After risk-adjustment, each hour of OT increased morbidity by 19%, UR by 8.7%, and LOS by 6.5 hours (all P < 0.001). For eLOS, procedures ≤5 hours had 9.5 times lower odds than ≥5 hours (P = 0.050), 5-7 hours had comparable odds (P = 0.540), and 7-9 hours had 5.5 times lower odds than procedures ≥ 9 hours (P < 0.001). Last, a multivariate linear regression showed that LOS can be calculated from OT: LOS (days) =1.527 + 0.272 × OT (hours) (R2 = 0.308; P < 0.001). Conclusions: OT (per hour) independently predicts morbidity, UR and LOS in DIEP flaps. Furthermore, 5 and 9 hours are critical cutoffs for eLOS. These findings emphasize the benefits of decreasing OT through efficiency models, such as process analysis, team-based intraoperative protocols, and co-surgery model.

Surface-induced phase separation of a sphingomyelin/cholesterol/ganglioside GM1-planar bilayer on mica surfaces and microdomain molecular conformation that accelerates Abeta oligomerization.

Ganglioside GM1 mediates the amyloid beta (Abeta) aggregation that is the hallmark of Alzheimer's disease (AD). To investigate how ganglioside-containing lipid bilayers interact with Abeta, we examined the interaction between Abeta40 and supported planar lipid bilayers (SPBs) on mica and SiO(2) substrates by using atomic force microscopy, fluorescence microscopy, and molecular dynamics computer simulations. These SPBs contained several compositions of sphingomyelin, cholesterol, and GM1 and were treated at physiological salt concentrations. Surprisingly high-speed Abeta aggregation of fibril formations occurred at all GM1 concentrations examined on the mica surface, but on the SiO(2) surface, only globular agglomerates formed and they formed slowly. At a GM1 concentration of 20mol%, unique triangular regions formed on the mica surface and the rapidly formed Abeta aggregations were observed only outside these regions. We have found that some unique surface-induced phase separations are induced by the GM1 clustering effects and the strong interactions between the GM1 head group and the water layer adsorbed in the ditrigonal cavities on the mica surface. The speed of Abeta40 aggregation and the shape of the agglomerates depend on the molecular conformation of GM1, which varies depending on the substrate materials. We identified the conformation that significantly accelerates Abeta40 aggregation, and we think that the detailed knowledge about the GM1 molecular conformation obtained in this work will be useful to those investigating Abeta-GM1 interactions.

Vertical Abdominoplasty Technique and the Impact of Preoperative Comorbidities on Outcomes.

BACKGROUND: As the number of patients requiring bariatric surgery has increased, so has the demand for body contouring after massive weight loss. Surgery involving the abdomen in these patients is particularly challenging as both vertical and horizontal laxity if often present, making traditional abdominoplasty techniques less effective. OBJECTIVES: The aim of this study was to review the operative technique and evaluate the preoperative comorbidities and operative decisions that may impact patient outcomes in those undergoing vertical abdominoplasty. METHODS: A review of the authors' technique is described. A retrospective chart review of patients who underwent vertical abdominoplasty for significant vertical and horizontal laxity after massive weight loss by a single surgeon between June 2007 and July 2019 was performed. Preoperative parameters, operative factors, and minor and major complications were evaluated. RESULTS: Our complication rate was 81% (13/16), which were all minor. No major complications were encountered. Patients with a history of nicotine use had a 100% complication rate. There was a trend toward higher seroma formation in patients with coronary artery disease and those with a history of smoking. There was also a trend toward higher wound dehiscence in patients with renal disease. The authors found no statistically significant correlation between complications and older age, higher weight of tissue resected, higher body mass index, and medical comorbidities. CONCLUSIONS: This small series helps to elucidate the role of vertical abdominoplasty in the care of patients following massive weight loss and its associated morbidity. Proper patient selection, appropriate preoperative patient counseling, and sound surgical technique help to mitigate the negative outcomes.

An asymmetric sheath controls flagellar supercoiling and motility in the leptospira spirochete.

Spirochete bacteria, including important pathogens, exhibit a distinctive means of swimming via undulations of the entire cell. Motility is powered by the rotation of supercoiled 'endoflagella' that wrap around the cell body, confined within the periplasmic space. To investigate the structural basis of flagellar supercoiling, which is critical for motility, we determined the structure of native flagellar filaments from the spirochete Leptospira by integrating high-resolution cryo-electron tomography and X-ray crystallography. We show that these filaments are coated by a highly asymmetric, multi-component sheath layer, contrasting with flagellin-only homopolymers previously observed in exoflagellated bacteria. Distinct sheath proteins localize to the filament inner and outer curvatures to define the supercoiling geometry, explaining a key functional attribute of this spirochete flagellum.

Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus.

Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild-type Chlamydomonas with CA mutants. We identified a large (>2 MD) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and 3D structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.

FcpB Is a Surface Filament Protein of the Endoflagellum Required for the Motility of the Spirochete Leptospira.

The spirochete endoflagellum is a unique motility apparatus among bacteria. Despite its critical importance for pathogenesis, the full composition of the flagellum remains to be determined. We have recently reported that FcpA is a novel flagellar protein and a major component of the sheath of the filament of the spirochete Leptospira. By screening a library of random transposon mutants in the spirochete Leptospira biflexa, we found a motility-deficient mutant harboring a disruption in a hypothetical gene of unknown function. Here, we show that this gene encodes a surface component of the endoflagellar filament and is required for typical hook- and spiral-shaped ends of the cell body, coiled structure of the endoflagella, and high velocity phenotype. We therefore named the gene fcpB for flagellar-coiling protein B. fcpB is conserved in all members of the Leptospira genus, but not present in other organisms including other spirochetes. Complementation of the fcpB- mutant restored the wild-type morphology and motility phenotypes. Immunoblotting with anti-FcpA and anti-FcpB antisera and cryo-electron microscopy of the filament indicated that FcpB assembled onto the surface of the sheath of the filament and mostly located on the outer (convex) side of the coiled filament. We provide evidence that FcpB, together with FcpA, are Leptospira-specific novel components of the sheath of the filament, key determinants of the coiled and asymmetric structure of the endoflagella and are essential for high velocity. Defining the components of the endoflagella and their functions in these atypical bacteria should greatly enhance our understanding of the mechanisms by which these bacteria produce motility.

Structural basis of cooperativity in kinesin revealed by 3D reconstruction of a two-head-bound state on microtubules.

The detailed basis of walking by dimeric molecules of kinesin along microtubules has remained unclear, partly because available structural methods have been unable to capture microtubule-bound intermediates of this process. Utilizing novel electron cryomicroscopy methods, we solved structures of microtubule-attached, dimeric kinesin bound to an ATP analog. We find that under these conditions, the kinesin dimer can attach to the microtubule with either one or two motor domains, and we present sub-nanometer resolution reconstructions of both states. The former structure reveals a novel kinesin conformation that revises the current understanding of how ATP binding is coupled to forward stepping of the motor. The latter structure indicates how tension between the two motor domains keeps their cycles out of phase in order to stimulate directional motility. The methods presented here pave the way for future structural studies of a variety of challenging macromolecules that bind to microtubules and other filaments.

[Vibrational spectra of thymine calculated from DFT and their comparison with experimental results].

The vibrational spectra of thymine were predicted by density functional theory (DFT). In the calculation B3LYP complex function, and diffuse function and polarization function were added to heavy atoms and light atoms. Taking the effects of H-bonds into consideration, the authors added two water molecules in our calculation and simulated the case of forming H-bonds in crystal thymine. Whilst the Raman and infrared spectra of thymine were recorded, and the spectra of thymine calculated from DFT agree with the experimental results. The calculation results in the present paper are more approximate to the experimental data than ever reported. The vibrational modes of thymine were assigned.

High-resolution structures of kinesin on microtubules provide a basis for nucleotide-gated force-generation.

Microtubule-based transport by the kinesin motors, powered by ATP hydrolysis, is essential for a wide range of vital processes in eukaryotes. We obtained insight into this process by developing atomic models for no-nucleotide and ATP states of the monomeric kinesin motor domain on microtubules from cryo-EM reconstructions at 5-6 Å resolution. By comparing these models with existing X-ray structures of ADP-bound kinesin, we infer a mechanistic scheme in which microtubule attachment, mediated by a universally conserved 'linchpin' residue in kinesin (N255), triggers a clamshell opening of the nucleotide cleft and accompanying release of ADP. Binding of ATP re-closes the cleft in a manner that tightly couples to translocation of cargo, via kinesin's 'neck linker' element. These structural transitions are reminiscent of the analogous nucleotide-exchange steps in the myosin and F1-ATPase motors and inform how the two heads of a kinesin dimer 'gate' each other to promote coordinated stepping along microtubules.