A cytokinetic ring-driven cell rotation achieves Hertwig's rule in early development.

Hertwig's rule states that cells divide along their longest axis, usually driven by forces acting on the mitotic spindle. Here, we show that in contrast to this rule, microtubule-based pulling forces in early Caenorhabditis elegans embryos align the spindle with the short axis of the cell. We combine theory with experiments to reveal that in order to correct this misalignment, inward forces generated by the constricting cytokinetic ring rotate the entire cell until the spindle is aligned with the cell's long axis. Experiments with slightly compressed mouse zygotes indicate that this cytokinetic ring-driven mechanism of ensuring Hertwig's rule is general for cells capable of rotating inside a confining shell, a scenario that applies to early cell divisions of many systems.

Signaling proteins in HSC fate determination are unequally segregated during asymmetric cell division.

Hematopoietic stem cells (HSCs) continuously replenish mature blood cells with limited lifespans. To maintain the HSC compartment while ensuring output of differentiated cells, HSCs undergo asymmetric cell division (ACD), generating two daughter cells with different fates: one will proliferate and give rise to the differentiated cells' progeny, and one will return to quiescence to maintain the HSC compartment. A balance between MEK/ERK and mTORC1 pathways is needed to ensure HSC homeostasis. Here, we show that activation of these pathways is spatially segregated in premitotic HSCs and unequally inherited during ACD. A combination of genetic and chemical perturbations shows that an ERK-dependent mechanism determines the balance between pathways affecting polarity, proliferation, and metabolism, and thus determines the frequency of asymmetrically dividing HSCs. Our data identify druggable targets that modulate HSC fate determination at the level of asymmetric division.

A Comparative Study on Survivors Versus Non-Survivors Among Diabetic Patients Having Mucormycosis.

Introduction Mucormycosis is a rare opportunistic fungal infection caused by fungi belonging to the Mucorales order and Mucoraceae family. It ranks as the third most prevalent angioinvasive fungal infection, following aspergillosis and candidiasis. This severe infection typically affects individuals with compromised immune systems, including those with hematological malignancies like leukemia and lymphoma, individuals who have undergone stem cell transplants, and people with diabetes mellitus. Individuals in good health are rarely affected, making immunocompromised individuals particularly vulnerable to this potentially fatal fungal disease. The aim of this study was to perform a comparative analysis of survivors versus non-survivors among diabetes patients admitted with mucormycosis. Methodology This was a descriptive observational study. A total of 338 patients were enrolled in the study. The study variables included demographics, COVID-19 infection, diabetes mellitus history, steroid use, use of oxygen support, and steam inhalation. Results Of the total 338 patients enrolled in the study, 253 (74.9%) were male and 85 (25.01%) were female. The number of survivors were 305 (90.2%) and non-survivors were 33(9.8%). The mean age of survivors was 52.50 ± 11.31 and non-survivors was 54.06 ± 8.54 years. Patients who underwent steam inhalation had a higher chance of survival compared to those who did not undergo steam inhalation and this association was statistically significant (p=0.01). Males showed a higher chance of survival (93.7%) as compared to females (80.0%). The associations between oxygen support, steroid use, and COVID-19 infection with the survival status were statistically non-significant. Conclusion There was a strong association between the history of steam inhalation and the outcome of mucormycosis among diabetes patients admitted with mucormycosis. Female patients demonstrated a higher fatality rate than males indicating a significant gender disparity observed in cases. Our findings may help to better identify and treat patients who are at higher risk for severe forms of mucormycosis.

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left-right symmetry breaking.

Proper left-right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left-right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left-right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin-dependent manner. Altogether, we conclude that CYK-1/Formin-dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left-right symmetry breaking in the nematode worm.

Regionalized tissue fluidization is required for epithelial gap closure during insect gastrulation.

Many animal embryos pull and close an epithelial sheet around the ellipsoidal egg surface during a gastrulation process known as epiboly. The ovoidal geometry dictates that the epithelial sheet first expands and subsequently compacts. Moreover, the spreading epithelium is mechanically stressed and this stress needs to be released. Here we show that during extraembryonic tissue (serosa) epiboly in the insect Tribolium castaneum, the non-proliferative serosa becomes regionalized into a solid-like dorsal region with larger non-rearranging cells, and a more fluid-like ventral region surrounding the leading edge with smaller cells undergoing intercalations. Our results suggest that a heterogeneous actomyosin cable contributes to the fluidization of the leading edge by driving sequential eviction and intercalation of individual cells away from the serosa margin. Since this developmental solution utilized during epiboly resembles the mechanism of wound healing, we propose actomyosin cable-driven local tissue fluidization as a conserved morphogenetic module for closure of epithelial gaps.

Cell lineage-dependent chiral actomyosin flows drive cellular rearrangements in early Caenorhabditis elegans development.

Proper positioning of cells is essential for many aspects of development. Daughter cell positions can be specified via orienting the cell division axis during cytokinesis. Rotatory actomyosin flows during division have been implied in specifying and reorienting the cell division axis, but how general such reorientation events are, and how they are controlled, remains unclear. We followed the first nine divisions of Caenorhabditis elegans embryo development and demonstrate that chiral counter-rotating flows arise systematically in early AB lineage, but not in early P/EMS lineage cell divisions. Combining our experiments with thin film active chiral fluid theory we identify a mechanism by which chiral counter-rotating actomyosin flows arise in the AB lineage only, and show that they drive lineage-specific spindle skew and cell reorientation events. In conclusion, our work sheds light on the physical processes that underlie chiral morphogenesis in early development.

Development of an innovative intrarenal pressure regulation system for mini-PCNL: A preliminary study.

INTRODUCTION: Miniaturized percutaneous nephrolithotomy (mini-PCNL) requires saline irrigation at high-pressures to maintain visual clarity. However, this may raise the intrarenal pelvic pressures (IRPs) beyond a safe range and may result in a higher complication rate. The aim of this study was to make and validate an automated pressure saline irrigation system to regulate IRPs during mini-PCNL. MATERIALS AND METHODS: A ureteric catheter was connected to an urodynamic machine and the minimum, maximum, and average IRPs reached during a standard 15 Fr mini-PCNL were measured in ten cases. Next, an intrarenal pressure regulation system (IPRS) was conceptualized, designed, patented, and constructed. IPRS was then tested on a mannequin model using the routine instruments. Lastly, the IPRS was evaluated on - five cases of 15 Fr mini-PCNL. The mean maximum IRP as recorded in the baseline data was set as the maximum permissible pressure on IPRS. The efficacy of IPRS was assessed by measuring the IRP, recorded in parallel, on both the IPRS and the urodynamic machine at various stages of the procedure. RESULTS: The mean maximum IRP reached during baseline evaluation was 25 cm of water which was set as the maximum permissible limit of the IPRS. Evaluation of the IRPS on mannequin models and validation clinical cases showed that IPRS measured the IRP accurately and prevented the pressure surge above the set limits Overall, higher IRPs were recorded during stone pulverization as compared to the other surgical steps. CONCLUSIONS: The current IPRS is the first of its kind open platform, portable, automated pressure saline irrigation system. It precisely monitors and controls the IRP and has the potential to reduce the irrigation pressure-related complications.

A novel paraboloid intracorporeal lithotripter: computer aided design analysis and in vitro comparison with holmium laser for large renal calculi.

PURPOSE: An intracorporeal lithotripsy probe tip was designed with a paraboloid shaped tip and compared with holmium laser for stone pulverization. MATERIALS AND METHODS: The paraboloid tip concept was developed and designed using computer aided design (CAD), fabricated, and patented. CAD analysis and in vitro comparison (with laser) of pulverization and propulsion dynamics were performed in an underwater hands-free bench arrangement using phantom stones. SPSS analysis for different energy cohorts was performed. RESULTS: CAD analysis: At "point contact" with the tip, the paraboloid lithotripter generated 3590 bars at generator settings of 4 bars. During "follow-up impacts," the tip pressure exponentially decreased (graduated tip pressure) and the lateral/centrifugal forces increased, converting the probe into a side-firing energy source. Bench analysis: At point contact, the paraboloid lithotripter at 2, 3, and 4 bars was comparable to that of a 6, 10, and 15 W laser, respectively (P<0.005). The paraboloid lithotripter showed a statistically significant advantage in breaking the phantoms, as against a laser that always bored through the phantom. Stone propulsion was comparable within all energy cohorts (P>0.05). CONCLUSION: The paraboloid lithotripter generates highly focused impact force with low propulsion, at point contact. As stone pulverization progresses, the tip forces exponentially decrease and the probe converts into a lateral firing energy source resulting in pulverization into larger fragments. Thus, the paraboloid lithotripter has all the advantages of laser at point contact and advantages of pneumatic lithotripter at follow-up hits, akin to being a bimodal energy source.

Neck dissection followed by definitive radiotherapy for small upper aerodigestive tract squamous cell carcinoma, with advanced neck disease: an alternative treatment strategy.

Treatment options for patients with small upper aerodigestive tracts squamous cell carcinoma (T1, T2) with advanced neck disease (N2, N3) is a topic that generates controversy in terms of thereuptic stratagies. We present the retrospective analysis of 109 patients treated, between 1991 and 2008, by "Neck dissection first approach" for N2, N3 neck node, followed by external beam radiotherapy (RT) with or without chemotherapy for the operated neck and the primary, deemed radiocurable. 94 patients completed the planned treatment and formed the material for this study. The primary (tumor) stage was as follows: T1 (29) and T2 (65) commonly arising from oropharynx; the neck nodes were predominantly N2a (n = 54), followed by N2b (n = 26) and N3 (n = 14) disease. Complete nodal clearence was achieved in 89 patients, with no major post operative complications. With a median follow up of 24 months disease free survival of 70% and overall survival of 61% at 5 years. Recurrence at primary site was noted predominantly with pyriform fossa tumors (n = 8), followed by base of tongue (n = 5) and were T2 lesions. Failure in the neck was seen in predominantly N3 nodes, R1 resection and failure to comply with adjuvant treatment. Neck dissection first approach is a valid treatment option that allows a good control of the disease in the neck, where it often fails if only RT is administered, along with preserving the pharyngolaryngeal function. Care should be excercised so that there should be no delay in initiating the RT following surgery.

Peptide truncation leads to a twist and an unusual increase in affinity for casitas B-lineage lymphoma tyrosine kinase binding domain.

We describe truncation and SAR studies to identify a pentapeptide that binds Cbl tyrosine kinase binding domain with a higher affinity than the parental peptide. The pentapeptide has an alternative binding mode that allows occupancy of a previously uncharacterized groove. A peptide library was used to map the binding site and define the interface landscape. Our results suggest that the pentapeptide is an ideal starting point for the development of inhibitors against Cbl driven diseases.

In vitro assessment of a novel spearheaded pneumatic intracorporeal lithotriptor.

BACKGROUND AND PURPOSE: Intracorporeal lithotripsy is an important modality used for stone pulverization. To improve the pulverization properties of intracorporeal lithotriptors, a novel intracorporeal "spearheaded lithotriptor" was designed by our institute. It was compared in vitro with the conventional lithotriptor. MATERIALS AND METHODS: The pulverization and propulsion dynamics were evaluated at various pressure settings on an in vitro bench arrangement with phantom stones. Lateral displacement during pulverization was also compared. RESULTS: The spearheaded lithotriptor had a better first hit (P<0.001) and follow-up hit dynamics (P<0.01). Stone propulsion and lateral displacement were low for the spearheaded lithotriptor at all pressure settings (P<0.05). CONCLUSION: The spearheaded lithotriptor improved stone pulverization without increasing the risk of stone migration. Further clinical evaluation of this novel probe is necessary.

High-throughput fluorescence polarization assay to identify inhibitors of Cbl(TKB)-protein tyrosine kinase interactions.

The casitas B-lineage lymphoma (Cbl) proteins play an important role in regulating signal transduction pathways by functioning as E3 ubiquitin ligases. The Cbl proteins contain a conserved tyrosine kinase binding (TKB) domain that binds more than a dozen proteins, including protein tyrosine kinases (PTKs), in a phosphorylation-dependent manner. The cell surface expression levels of the PTKs are regulated by Cbl-mediated ubiquitination, internalization, and degradation. Dysfunction in this signaling cascade has resulted in prolonged activation of the PTKs and, therefore, has been implicated in inflammatory diseases and various cancers. Due to this negative regulatory function, Cbl has been largely ignored as a therapeutic target. However, recent studies, such as the identification of (i) gain of function c-Cbl mutations in subsets of myeloid cancer and (ii) c-Cbl as a prostate basal cell marker that correlates with poor clinical outcome, suggest otherwise. Here we report the development of a competitive high-throughput fluorescence polarization assay in a 384-well format to identify inhibitors of Cbl(TKB). The high-throughput screen readiness of the assay was demonstrated by screening the Prestwick Chemical Library.

Structure of a Venezuelan equine encephalitis virus assembly intermediate isolated from infected cells.

Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.

Structural characterization of BRCT-tetrapeptide binding interactions.

BRCT(BRCA1) plays a major role in DNA repair pathway, and does so by recognizing the conserved sequence pSXXF in its target proteins. Remarkably, tetrapeptides containing pSXXF motif bind with high specificity and micromolar affinity. Here, we have characterized the binding interactions of pSXXF tetrapeptides using NMR spectroscopy and calorimetry. We show that BRCT is dynamic and becomes structured on binding, that pSer and Phe residues dictate overall binding, and that the binding affinities of the tetrapeptides are intimately linked to structural and dynamic changes both in the BRCT(BRCA1) and tetrapeptides. These results provide critical insights for designing high-affinity BRCT(BRCA1) inhibitors.

Dual-fluorophore quantitative high-throughput screen for inhibitors of BRCT-phosphoprotein interaction.

Finding specific small-molecule inhibitors of protein-protein interactions remains a significant challenge. Recently, attention has grown toward "hot spot" interactions where binding is dominated by a limited number of amino acid contacts, theoretically offering an increased opportunity for disruption by small molecules. Inhibitors of the interaction between BRCT (the C-terminal portion of BRCA1, a key tumor suppressor protein with various functions) and phosphorylated proteins (Abraxas/BACH1/CtIP), implicated in DNA damage response and repair pathways, should prove to be useful in studying BRCA1's role in cancer and in potentially sensitizing tumors to chemotherapeutic agents. We developed and miniaturized to a 1536-well format and 3-mul final volume a pair of fluorescence polarization (FP) assays using fluorescein- and rhodamine-labeled pBACH1 fragment. To minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the 75,552 compound library members all were assayed against both the fluorescein- and rhodamine-labeled probe-protein complexes in separate but interleaved reactions. In addition, every library compound was tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm. Analyses of the screening results led to the selection and subsequent confirmation of 16 compounds active in both assays. Faced with a traditionally difficult protein-protein interaction assay, by performing two-fluorophore qHTS, we were able to confidently select a number of actives for further studies.

High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1.

The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G(2) phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)-BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide (BDP1) containing the critical phosphoserine, a phenylalanine at (P+3), and a GST-BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant (K(d)) of 1.58+/-0.01microM and a dynamic range (DeltamP) of 164.9+/-1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z' factor of 0.87 was achieved in a 384-well format for high-throughput screening.

Structural studies on recombinant T = 3 capsids of Sesbania mosaic virus coat protein mutants.

When expressed in Escherichia coli, the recombinant coat protein (rCP) of Sesbania mosaic virus (SeMV) was shown to self-assemble into T = 3 capsids encapsidating CP mRNA and 23S rRNA derived from the host. Expression of CP-P53A, in which a conserved proline at position 53 in the beta-annulus was substituted by alanine (CP-P53A), also produced similar capsids. Purified rCP and CP-P53A particles were crystallized and X-ray crystal structures of their mutant capsids were determined to resolutions of 3.6 and 4.1 A, respectively. As in the native viral CP, the CPs in these recombinant capsids adopt the jelly-roll beta-sandwich fold. The amino-terminal residues of the C subunits alone are ordered and form the beta-annulus structure at the quasi-sixfold axes. A characteristic bend in the beta-annulus remains unaffected in CP-P53A. The quasi-threefold interfaces of the capsids harbour calcium ions coordinated by ligands from the adjacent threefold-related subunits in a geometry that is analogous to that observed in the native capsid. Taken together with studies on deletion and substitution mutants of SeMV CP, these results suggest the possibility that the beta-annulus and nucleic acid-mediated interactions may be less important for the assembly of sobemoviruses than previously envisaged.

The role of arginine-rich motif and beta-annulus in the assembly and stability of Sesbania mosaic virus capsids.

Sesbania mosaic virus (SeMV) capsids are stabilized by protein-protein, protein-RNA and calcium-mediated protein-protein interactions. The N-terminal random domain of SeMV coat protein (CP) controls RNA encapsidation and size of the capsids and has two important motifs, the arginine-rich motif (ARM) and the beta-annulus structure. Here, mutational analysis of the arginine residues present in the ARM to glutamic acid was carried out. Mutation of all the arginine residues in the ARM almost completely abolished RNA encapsidation, although the assembly of T=3 capsids was not affected. A minimum of three arginine residues was found to be essential for RNA encapsidation. The mutant capsids devoid of RNA were less stable to thermal denaturation when compared to wild-type capsids. The results suggest that capsid assembly is entirely mediated by CP-dependent protein-protein inter-subunit interactions and encapsidation of genomic RNA enhances the stability of the capsids. Because of the unique structural ordering of beta-annulus segment at the icosahedral 3-folds, it has been suggested as the switch that determines the pentameric and hexameric clustering of CP subunits essential for T=3 capsid assembly. Surprisingly, mutation of a conserved proline within the segment that forms the beta-annulus to alanine, or deletion of residues 48-53 involved in hydrogen bonding interactions with residues 54-58 of the 3-fold related subunit or deletion of all the residues (48-59) involved in the formation of beta-annulus did not affect capsid assembly. These results suggest that the switch for assembly into T=3 capsids is not the beta-annulus. The ordered beta-annulus observed in the structures of many viruses could be a consequence of assembly to optimize intersubunit interactions.

T=1 capsid structures of Sesbania mosaic virus coat protein mutants: determinants of T=3 and T=1 capsid assembly.

Sesbania mosaic virus particles consist of 180 coat protein subunits of 29kDa organized on a T=3 icosahedral lattice. N-terminal deletion mutants of coat protein that lack 36 (CP-NDelta36) and 65 (CP-NDelta65) residues from the N terminus, when expressed in Escherichia coli, produced similar T=1 capsids of approximate diameter 20nm. In contrast to the wild-type particles, these contain only 60 copies of the truncated protein subunits (T=1). CP-NDelta65 lacks the "beta-annulus" believed to be responsible for the error-free assembly of T=3 particles. Though the CP-NDelta36 mutant has the beta-annulus segment, it does not form a T=3 capsid, presumably because it lacks an arginine-rich motif found close to the amino terminus. Both CP-NDelta36 and CP-NDelta65 T=1 capsids retain many key features of the T=3 quaternary structure. Calcium binding geometries at the coat protein interfaces in these two particles are also nearly identical. When the conserved aspartate residues that coordinate the calcium, D146 and D149 in the CP-NDelta65, were mutated to asparagine (CP-NDelta65-D146N-D149N), the subunits assembled into T=1 particles but failed to bind calcium ions. The structure of this mutant revealed particles that were slightly expanded. The analysis of the structures of these mutant capsids suggests that although calcium binding contributes substantially to the stability of T=1 particles, it is not mandatory for their assembly. In contrast, the presence of a large fraction of the amino-terminal arm including sequences that precede the beta-annulus and the conserved D149 appear to be indispensable for the error-free assembly of T=3 particles.