Analysis of the Architecture of the Nuclear Pore Complex by 3D super-resolution fluorescence microscopy

Nuclear pore complexes (NPCs), embedded in the two nuclear membranes, are the unique gateways that mediate all the traffic between the nucleus and the cytoplasm. In higher eukaryotes, each NPC is composed of multiple copies of approximately 30 different proteins termed nucleoporins and has a mass of over 100 MDa. In recent years, substantial effort has been devoted to the structural and functional characterization of this essential molecular machine in eukaryotic cells. Even though a pseudo-atomic model of the scaffold of the NPC has been produced, many details of the structure still remain elusive due to the enormous size, complexity and conformational dynamics of the NPC. In addition, we have little structural understanding of how such a large machine is assembled and what dynamic structural changes underlie its functions. During my PhD work, I established a methodology that can determine the 3D architecture of the NPC by combining single-molecule localization microscopy in a 4Pi detection scheme with computational classification and 3D single particle averaging. This new approach is able to resolve the structure of the NPC with molecular specificity and nano-scale resolution in situ in human cells. I here present the reconstruction of a 3D molecular map that integrates both scaffold and flexible nucleoporins and that allows us to address dynamic components of the NPC that have been inaccessible for atomic resolution methods to date. My findings indicate that the peripheral regions of the NPC can assume very different conformational states and that even the overall scaffold structure of the NPC is more flexible than previously assumed. The methodology I have established opens the exciting possibility to address novel structural, functional and assembly aspects of this fundamental cellular machine and can be applied to interrogate the 3D architecture of other large protein complexes and organelles inside cells

Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically-Encoded Probes for DNA-PAINT

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super-resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of circa 3 nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy

The Growth of CdTe Layer on GaAs Substrate by MBE

We present the results of growth of CdTe layer on (013)GaAs substrate. The sequence processes include the preparation of GaAs surface by chemical etching and annealing in ultra-high vacuum, the growth of ZnTe layer on atomically clean GaAs surface and then the growth of CdTe layer on ZnTe/GaAs. All processes were carried out without removing GaAs substrate from MBE set. The processes were controlled by RHEED and single wavelength ellipsometry. We found that the evaporation of arsenic oxides and gallium oxides from the (001)GaAs surface were observed at over 400 and 500°C, respectively. The growth of CdTe on (001)GaAs leads to appearance of mixture orientations because of large mismatch of lattice parameters. We study the growth of ZnTe on (001)GaAs and (013)GaAs substrates to prevent the growth of mixture orientations. We study the influence of cadmium and tellurium ratio in molecular fluxes and temperature on the growth mechanism of ZnTe and CdTe, crystal perfection, surface roughness and defects density. The optimal condition for growth of high quality thick CdTe on GaAs substrate were found

Nonlinear diffusion equations as asymptotic limits of Cahn-Hilliard systems

An asymptotic limit of a class of Cahn-Hilliard systems is investigated to obtain a general nonlinear diffusion equation. The target diffusion equation may reproduce a number of well-known model equations: Stefan problem, porous media equation, Hele-Shaw profile, nonlinear diffusion of singular logarithmic type, nonlinear diffusion of Penrose-Fife type, fast diffusion equation and so on. Namely, by setting the suitable potential of the Cahn-Hilliard systems, all of these problems can be obtained as limits of the Cahn-Hilliard related problems. Convergence results and error estimates are proved

Sperm chemotaxis is driven by the slope of the chemoattractant concentration field.

Spermatozoa of marine invertebrates are attracted to their conspecific female gamete by diffusive molecules, called chemoattractants, released from the egg investments in a process known as chemotaxis. The information from the egg chemoattractant concentration field is decoded into intracellular Ca2+ concentration ([Ca2+]i) changes that regulate the internal motors that shape the flagellum as it beats. By studying sea urchin species-specific differences in sperm chemoattractant-receptor characteristics we show that receptor density constrains the steepness of the chemoattractant concentration gradient detectable by spermatozoa. Through analyzing different chemoattractant gradient forms, we demonstrate for the first time that Strongylocentrotus purpuratus sperm are chemotactic and this response is consistent with frequency entrainment of two coupled physiological oscillators: i) the stimulus function and ii) the [Ca2+]i changes. We demonstrate that the slope of the chemoattractant gradients provides the coupling force between both oscillators, arising as a fundamental requirement for sperm chemotaxis

ПЕРЕБІГ ВІЛ/ГС-КОІНФЕКЦІЇ В ПОЛТАВСЬКІЙ ОБЛАСТІ ЗАЛЕЖНО ВІД ШЛЯХІВ ПЕРЕДАЧІ

Analysis of HIV/HCV-coinfection in Poltava region, depending on the transmission of HIV. It was found that clinical stages III (p=0,038) and IV (p=0,044) were recorded significantly more often at the time of diagnosis of HIV infection among patients with parenteral route of transmission, whereas among patients with sexually transmission, by contrast, clinical stage I (p=0,001) was found more often.In patients with HIV/HCV-coinfection with parenteral route of HIV opportunistic infections (FDPT, oropharyngeal candidiasis, bacterial pneumonia) were often diagnosed at higher levels of CD4-lymphocyte (≥100 cells/ml). There is no difference between group of patients with significant immunodeficiency (level of CD4-lymphocytes <100 cells/microliter) and group of patients with HIV/HCV coinfection in identifying of important opportunistic infections.Проведено аналіз перебігу ВІЛ/ГС інфекції в пацієнтів Полтавської області залежно від шляхів передачі ВІЛ. Виявлено, що на момент встановлення діагнозу ВІЛ-інфекції серед пацієнтів з парентеральним шляхом передачі частіше реєстрували ІІІ (р=0,038) і ІV (р=0,044) клінічні стадії, в той час як серед пацієнтів із статевим шляхом передачі, навпаки, частіше виявляли І клінічну стадію (р=0,001).У ВІЛ/ГС коінфікованих осіб з парентеральним шляхом передачі ВІЛ опортуністичні інфекції вперше діагностований туберкульоз (ВДТБ), кандидоз орофарингеальний, бактерійну пневмонію) частіше діагностували при вищих рівнях СD4-лімфоцитів (≥100 кл./мкл). За наявності вираженого імунодефіциту (рівень СD4-лімфоцитів <100 кл./мкл) відмінностей між групами ВІЛ/ГС коінфікованих пацієнтів щодо виявлення значимих опортуністичних інфекцій не виявлено

An epigenetic switch regulates the ontogeny of AXL-positive/EGFR-TKi-resistant cells by modulating miR-335 expression.

Despite current advancements in research and therapeutics, lung cancer remains the leading cause of cancer-related mortality worldwide. This is mainly due to the resistance that patients develop against chemotherapeutic agents over the course of treatment. In the context of non-small cell lung cancers (NSCLC) harboring EGFR-oncogenic mutations, augmented levels of AXL and GAS6 have been found to drive resistance to EGFR tyrosine kinase inhibitors such as Erlotinib and Osimertinib in certain tumors with mesenchymal-like features. By studying the ontogeny of AXL-positive cells, we have identified a novel non-genetic mechanism of drug resistance based on cell-state transition. We demonstrate that AXL-positive cells are already present as a subpopulation of cancer cells in Erlotinib-naïve tumors and tumor-derived cell lines and that the expression of AXL is regulated through a stochastic mechanism centered on the epigenetic regulation of miR-335. The existence of a cell-intrinsic program through which AXL-positive/Erlotinib-resistant cells emerge infers the need of treating tumors harboring EGFR-oncogenic mutations upfront with combinatorial treatments targeting both AXL-negative and AXL-positive cancer cells