5-ALA-guided tumor resection during awake speech mapping in gliomas located in eloquent speech areas: Single-center experience.

Background: Achieving maximal functionally safe resection of gliomas located within the eloquent speech areas is challenging, and there is a lack of literature on the combined use of 5-aminolevulinic acid (5-ALA) guidance and awake craniotomy. Objective: The aim of this study was to describe our experience with the simultaneous use of 5-ALA fluorescence and awake speech mapping in patients with left frontal gliomas located within the vicinity of eloquent speech areas. Materials and methods: A prospectively collected database of patients was reviewed. 5-ALA was administered at a dose of 20 mg/kg 2 h prior to operation, and an operating microscope in BLUE400 mode was used to visualize fluorescence. All patients underwent surgery using the "asleep-awake-asleep" protocol with monopolar and bipolar electrical stimulation to identify the proximity of eloquent cortex and white matter tracts and to guide safe limits of resection along with fluorescence guidance. Speech function was assessed by a trained neuropsychologist before, during, and after surgery. Results: In 28 patients operated with cortical mapping and 5-ALA guidance (12 Grade 4, 6 Grade 3, and 10 Grade 2 gliomas), Broca's area was identified in 23 cases and Wernicke's area was identified in 5 cases. Fluorescence was present in 14 cases. Six tumors had residual fluorescence due to the positive speech mapping in the tumor bed. Transient aphasia developed in 14 patients, and permanent aphasia developed in 4 patients. In 6 patients operated with cortical and subcortical speech mapping and 5-ALA guidance (4 Grade 4, 1 Grade 3, and 1 Grade 2 gliomas), cortical speech areas were mapped in 5 patients and subcortical tracts were encountered in all cases. In all cases, resection was stopped despite the presence of residual fluorescence due to speech mapping findings. Transient aphasia developed in 6 patients and permanent aphasia developed in 4 patients. In patients with Grade 2-3 gliomas, targeted biopsy of focal fluorescence areas led to upgrading the grade and thus more accurate diagnosis. Conclusion: 5-ALA guidance during awake speech mapping is useful in augmenting the extent of resection for infiltrative high-grade gliomas and identifying foci of anaplasia in non-enhancing gliomas, while maintaining safe limits of functional resection based on speech mapping. Positive 5-ALA fluorescence in diffuse Grade 2 gliomas may be predictive of a more aggressive disease course.

Griottes: a generalist tool for network generation from segmented tissue images.

BACKGROUND: Microscopy techniques and image segmentation algorithms have improved dramatically this decade, leading to an ever increasing amount of biological images and a greater reliance on imaging to investigate biological questions. This has created a need for methods to extract the relevant information on the behaviors of cells and their interactions, while reducing the amount of computing power required to organize this information. RESULTS: This task can be performed by using a network representation in which the cells and their properties are encoded in the nodes, while the neighborhood interactions are encoded by the links. Here, we introduce Griottes, an open-source tool to build the "network twin" of 2D and 3D tissues from segmented microscopy images. We show how the library can provide a wide range of biologically relevant metrics on individual cells and their neighborhoods, with the objective of providing multi-scale biological insights. The library's capacities are demonstrated on different image and data types. CONCLUSIONS: This library is provided as an open-source tool that can be integrated into common image analysis workflows to increase their capacities.

Super-resolution visualization and modeling of human chromosomal regions reveals cohesin-dependent loop structures.

BACKGROUND: The 3D organization of the chromatin fiber in cell nuclei plays a key role in the regulation of gene expression. Genome-wide techniques to score DNA-DNA contacts, such as Hi-C, reveal the partitioning of chromosomes into epigenetically defined active and repressed compartments and smaller "topologically associated" domains. These domains are often associated with chromatin loops, which largely disappear upon removal of cohesin. Because most Hi-C implementations average contact frequencies over millions of cells and do not provide direct spatial information, it remains unclear whether and how frequently chromatin domains and loops exist in single cells. RESULTS: We combine 3D single-molecule localization microscopy with a low-cost fluorescence labeling strategy that does not denature the DNA, to visualize large portions of single human chromosomes in situ at high resolution. In parallel, we develop multi-scale, whole nucleus polymer simulations, that predict chromatin structures at scales ranging from 5 kb up to entire chromosomes. We image chromosomes in G1 and M phase and examine the effect of cohesin on interphase chromatin structure. Depletion of cohesin leads to increased prevalence of loose chromatin stretches, increased gyration radii, and reduced smoothness of imaged chromatin regions. By comparison to model predictions, we estimate that 6-25 or more purely cohesin-dependent chromatin loops coexist per megabase of DNA in single cells, suggesting that the vast majority of the genome is enclosed in loops. CONCLUSION: Our results provide new constraints on chromatin structure and showcase an affordable non-invasive approach to study genome organization in single cells.

The transpeptidase PBP2 governs initial localization and activity of the major cell-wall synthesis machinery in E. coli.

Bacterial shape is physically determined by the peptidoglycan cell wall. The cell-wall-synthesis machinery responsible for rod shape in Escherichia coli is the processive 'Rod complex'. Previously, cytoplasmic MreB filaments were thought to govern formation and localization of Rod complexes based on local cell-envelope curvature. Using single-particle tracking of the transpeptidase and Rod-complex component PBP2, we found that PBP2 binds to a substrate different from MreB. Depletion and localization experiments of other putative Rod-complex components provide evidence that none of those provide the sole rate-limiting substrate for PBP2 binding. Consistently, we found only weak correlations between MreB and envelope curvature in the cylindrical part of cells. Residual correlations do not require curvature-based Rod-complex initiation but can be attributed to persistent rotational motion. We therefore speculate that the local cell-wall architecture provides the cue for Rod-complex initiation, either through direct binding by PBP2 or through an unknown intermediate.

Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects.

Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

Perspectives and Consensus among International Orthopaedic Surgeons during Initial and Mid-lockdown Phases of Coronavirus Disease.

With a lot of uncertainty, unclear, and frequently changing management protocols, COVID-19 has significantly impacted the orthopaedic surgical practice during this pandemic crisis. Surgeons around the world needed closed introspection, contemplation, and prospective consensual recommendations for safe surgical practice and prevention of viral contamination. One hundred orthopaedic surgeons from 50 countries were sent a Google online form with a questionnaire explicating protocols for admission, surgeries, discharge, follow-up, relevant information affecting their surgical practices, difficulties faced, and many more important issues that happened during and after the lockdown. Ten surgeons critically construed and interpreted the data to form rationale guidelines and recommendations. Of the total, hand and microsurgery surgeons (52%), trauma surgeons (32%), joint replacement surgeons (20%), and arthroscopy surgeons (14%) actively participated in the survey. Surgeons from national public health care/government college hospitals (44%) and private/semiprivate practitioners (54%) were involved in the study. Countries had lockdown started as early as January 3, 2020 with the implementation of partial or complete lifting of lockdown in few countries while writing this article. Surgeons (58%) did not stop their surgical practice or clinics but preferred only emergency cases during the lockdown. Most of the surgeons (49%) had three-fourths reduction in their total patients turn-up and the remaining cases were managed by conservative (54%) methods. There was a 50 to 75% reduction in the number of surgeries. Surgeons did perform emergency procedures without COVID-19 tests but preferred reverse transcription polymerase chain reaction (RT-PCR; 77%) and computed tomography (CT) scan chest (12%) tests for all elective surgical cases. Open fracture and emergency procedures (60%) and distal radius (55%) fractures were the most commonly performed surgeries. Surgeons preferred full personal protection equipment kits (69%) with a respirator (N95/FFP3), but in the case of unavailability, they used surgical masks and normal gowns. Regional/local anesthesia (70%) remained their choice for surgery to prevent the aerosolized risk of contaminations. Essential surgical follow-up with limited persons and visits was encouraged by 70% of the surgeons, whereas teleconsultation and telerehabilitation by 30% of the surgeons. Despite the protective equipment, one-third of the surgeons were afraid of getting infected and 56% feared of infecting their near and dear ones. Orthopaedic surgeons in private practice did face 50 to 75% financial loss and have to furlough 25% staff and 50% paramedical persons. Orthopaedics meetings were cancelled, and virtual meetings have become the preferred mode of sharing the knowledge and experiences avoiding human contacts. Staying at home, reading, and writing manuscripts became more interesting and an interesting lifestyle change is seen among the surgeons. Unanimously and without any doubt all accepted the fact that COVID-19 pandemic has reached an unprecedented level where personal hygiene, hand washing, social distancing, and safe surgical practices are the viable antidotes, and they have all slowly integrated these practices into their lives. Strict adherence to local authority recommendations and guidelines, uniform and standardized norms for admission, inpatient, and discharge, mandatory RT-PCR tests before surgery and in selective cases with CT scan chest, optimizing and regularizing the surgeries, avoiding and delaying nonemergency surgeries and follow-up protocols, use of teleconsultations cautiously, and working in close association with the World Health Organization and national health care systems will provide a conducive and safe working environment for orthopaedic surgeons and their fraternity and also will prevent the resurgence of COVID-19.

ZOLA-3D allows flexible 3D localization microscopy over an adjustable axial range.

Single molecule localization microscopy can generate 3D super-resolution images without scanning by leveraging the axial variations of normal or engineered point spread functions (PSF). Successful implementation of these approaches for extended axial ranges remains, however, challenging. We present Zernike Optimized Localization Approach in 3D (ZOLA-3D), an easy-to-use computational and optical solution that achieves optimal resolution over a tunable axial range. We use ZOLA-3D to demonstrate 3D super-resolution imaging of mitochondria, nuclear pores and microtubules in entire nuclei or cells up to ~5 µm deep.

Deep learning massively accelerates super-resolution localization microscopy.

The speed of super-resolution microscopy methods based on single-molecule localization, for example, PALM and STORM, is limited by the need to record many thousands of frames with a small number of observed molecules in each. Here, we present ANNA-PALM, a computational strategy that uses artificial neural networks to reconstruct super-resolution views from sparse, rapidly acquired localization images and/or widefield images. Simulations and experimental imaging of microtubules, nuclear pores, and mitochondria show that high-quality, super-resolution images can be reconstructed from up to two orders of magnitude fewer frames than usually needed, without compromising spatial resolution. Super-resolution reconstructions are even possible from widefield images alone, though adding localization data improves image quality. We demonstrate super-resolution imaging of >1,000 fields of view containing >1,000 cells in ∼3 h, yielding an image spanning spatial scales from ∼20 nm to ∼2 mm. The drastic reduction in acquisition time and sample irradiation afforded by ANNA-PALM enables faster and gentler high-throughput and live-cell super-resolution imaging.

3D plasmonic crystal metamaterials for ultra-sensitive biosensing.

We explore the excitation of plasmons in 3D plasmon crystal metamaterials and report the observation of a delocalized plasmon mode, which provides extremely high spectral sensitivity (>2600 nm per refractive index unit (RIU) change), outperforming all plasmonic counterparts excited in 2D nanoscale geometries, as well as a prominent phase-sensitive response (>3*10(4) deg. of phase per RIU). Combined with a large surface for bioimmobilization provided by the 3D matrix, the proposed sensor architecture promises a new important landmark in the advancement of plasmonic biosensing technology.