Phasor Histone FLIM-FRET Microscopy Maps Nuclear-Wide Nanoscale Chromatin Architecture With Respect to Genetically Induced DNA Double-Strand Breaks

A DNA double-strand break (DSB) takes place in the context of chromatin, and there is increasing evidence for chromatin structure to play a functional role in DSB signaling and repair. Thus, there is an emerging need for quantitative microscopy methods that can directly measure chromatin network architecture and detect changes in this structural framework upon DSB induction within an intact nucleus. To address this demand, here we present the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labeled histones in the DSB inducible via AsiSI cell system (DIvA), which has sufficient spatial resolution to map nuclear-wide chromatin compaction at the level of nucleosome proximity with respect to multiple site-specific DSBs. We also demonstrate that when phasor histone FLIM-FRET is coupled with immunofluorescence, this technology has the unique advantage of enabling exploration of any heterogeneity that exists in chromatin structure at the spatially distinct and genetically induced DSBs.

Three-dimensional deconvolution processing for STEM cryotomography

The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.

The mitotic protein NuMA plays a spindle-independent role in nuclear formation and mechanics

Eukaryotic cells typically form a single, round nucleus after mitosis, and failures to do so can compromise genomic integrity. How mammalian cells form such a nucleus remains incompletely understood. NuMA is a spindle protein whose disruption results in nuclear fragmentation. What role NuMA plays in nuclear integrity, or whether its perceived role stems from its spindle function, is unclear. Here, we use live imaging to demonstrate that NuMA plays a spindle-independent role in forming a single, round nucleus. NuMA keeps the decondensing chromosome mass compact at mitotic exit, and promotes a mechanically robust nucleus. NuMA’s C-terminus binds DNA in vitro and chromosomes in interphase, while its coiled-coil acts as a regulatory and structural hub: it prevents NuMA from binding chromosomes at mitosis, regulates its nuclear mobility and is essential for nuclear formation. Thus, NuMA plays a long-range structural role in building and maintaining an intact nucleus, as it does for the spindle, playing a protective role over the cell cycle.

Closed mitosis requires local disassembly of the nuclear envelope

At the end of mitosis, eukaryotic cells must segregate both copies of their replicated genome into two new nuclear compartments (1). They do this either by first dismantling and later reassembling the nuclear envelope in a so called “open mitosis”, or by reshaping an intact nucleus and then dividing into two in a “closed mitosis” (2, 3). However, while mitosis has been studied in a wide variety of eukaryotes for over a century (4), it is not known how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment (5). In studying this problem in the fission yeast Schizosaccharomyces pombe, a classical model for closed mitosis (5), we use genetics, live cell imaging and electron tomography to show that nuclear fission is achieved via local disassembly of the nuclear envelope (NE) within the narrow bridge that links segregating daughter nuclei. In doing so, we identify a novel inner NE-localised protein Les1 that restricts the process of local NE breakdown (local NEB) to the bridge midzone and prevents the leakage of material from daughter nuclei. The mechanics of local NEB in a closed mitosis closely mirror those of NEB in open mitosis (3), revealing an unexpectedly deep conservation of nuclear remodelling mechanisms across diverse eukaryotes.

Circulating tumor cell (CTC) enumeration in patients (pts) with metastatic genitourinary (mGU) tumors treated in a phase I study of cabozantinib and nivolumab (CaboNivo) +/- ipilimumab (CaboNivoIpi).

4555 Background: CTCs may serve as biomarkers for clinical outcomes in GU tumor pts. We examined the association between baseline CTC enumeration, CTC heterogeneity, CTC morphologic subtypes, and progression-free-survival, overall survival and response to therapy with combination CaboNivo or CaboNivoIpi. Methods: 123 samples from 52 pts with mGU tumors treated with CaboNivo (38 pts) or CaboNivoIpi (14 pts) drawn at Baseline, Cycle (C) 2 Day (D) 1, and C3D1 were processed using the Epic Sciences platform. CTCs were defined as cytokeratin (CK)+, CD45-, distinct morphology, intact nucleus. PD-L1 expression was also assessed. Results: From 07/20/2016-09/01/2018, 52 pts [urothelial carcinoma (UC) N = 33; plasmacytoid N = 1; Clear cell renal cell carcinoma N = 4; bladder adenocarcinoma N = 8; bladder squamous cell carcinoma N = 2; bladder small cell N = 2; renal medullary N = 2] were treated. Median age was 61.5 years (range 20-82); 35 (67%) were male. N = 37 (71%) had visceral involvement, N = 15 (29%) with liver involvement, N = 11 (21%) with bone involvement. CTCs were found in the peripheral blood of 26/40 (65%) pts at baseline. 1/40 pts (bladder adenocarcinoma) had PDL1+ CTCs at Baseline. Median CTC/mL at Baseline, C2D1, C3D1, were not significantly different. CTC counts of > 2 and > 4 at C2D1 were potentially associated with shorter OS (p = 0.071 and p = 0.045 without adjustment for multiple cutoffs for evaluation). PFS results exhibited similar trends. Unsupervised clustering of CTC images identified 5 main CTC subtypes, of which the presence of one was significantly associated with shorter OS (p = 0.0014, not adjusted for multiple testing). Additionally, we observed a trend towards patients with higher CTC heterogeneity at C2D1 having longer OS, when adjusting for the risk associated with CTC enumeration (p = 0.084). Conclusions: CTC were detected in pts with mGU tumors treated with CaboNivo and CaboNivoIpi. CTC values were somewhat but not statistically lower in responders vs. non-responders. On treatment lower CTCs and the absence of aggressive CTC subtypes were associated with better clinical outcomes. Ongoing analyses include single cell genomics, and analysis of T-cell populations. Clinical trial information: NCT02496208.

Circulating tumor cell (CTC) enumeration in patients (pts) with metastatic genitourinary (mGU) tumors treated in a phase I study of cabozantinib and nivolumab (CaboNivo) +/- ipilimumab (CaboNivoIpi).

379 Background: CTCs may serve as biomarkers for clinical outcomes in GU tumor pts. We examined the association between baseline CTC enumeration, change in CTC enumeration post treatment, progression-free-survival, overall survival and response to therapy with combination CaboNivo or CaboNivoIpi. Methods: 128 samples from 52 pts with mGU tumors treated with CaboNivo (38 pts) or CaboNivoIpi (14 pts) were drawn at baseline, cycle (C) 2 Day (D) 1, and C3D1 and processed with Epic Sciences platform. CTCs were defined as cytokeratin (CK)+, CD45-, distinct morphology, intact nucleus. PD-L1 expression was assessed. Results: From 07/20/2016-09/01/2018, 52 pts were treated for metastatic [urothelial carcinoma (UC) N = 33; UC plasmacytoid N = 1; clear cell renal cell carcinoma N = 4; bladder adenocarcinoma (BAC) N = 8; bladder squamous cell carcinoma N = 2; bladder small cell carcinoma N = 2; renal medullary carcinoma N = 2]. Median age was 61.5 years (range 20-82); 35 (67%) were male; N = 37(71%) had visceral involvement, N = 15(29%) had liver involvement, N = 11(21%) had bone involvement. CTCs were found in the peripheral blood of 39 (75%) pts, 1 pt (BAC) had PDL1+ CTCs at baseline. Median CTC/mL at C1D1, C2D1, C3D1, were 0.5 (0-357.1), 0.4 (0-71.8), and 0.75 (0-18), respectively. Median (lower and upper quartile) CTCs in pts with complete or partial responses (responders) vs stable or progressive disease (non-responders) were: 0.0 (0 to 5.40) vs 0.7 (0 to 2.5) at baseline; -0.35 (-3.65 to 0) vs 0 (-2 to 1.7) change from baseline to C2D1; and 0 (-5.4 to 0.5) vs 0 (-0.9 to 0.65) change from baseline to C3D1 (all p > 0.15). CTC counts of > 2 and > 4 at C2D1 were potentially associated with shorter OS (p = 0.071 and p = 0.045 without adjustment for multiple cutoffs for evaluation). PFS results exhibited similar trends. Conclusions: CTC were detected in pts with mGU tumors treated with CaboNivo and CaboNivoIpi. CTC values were lower in responders than in non-responders. On treatment lower CTCs were associated with better clinical outcomes. Ongoing analyses with the CTCs include cell morphology and heterogeneity, single cell genomics, and T-cell populations.

Clinical and morphological assessment of low frequency ultrasound use in delayed eruption of teeth

Aim. Development of a new method stimulating eruption of impacted teeth, reducing side effects and the term of orthodontic treatment. Methods. Considering the high numbers of patients who sought medical aid with delayed eruption of teeth, rational method of low-frequency ultrasound was used for stimulating eruption of impacted teeth. The stimulation process was performed by «SIAZ-EGS Med-Stomo» device, with the oscillation frequency of 26.5 kHz, intensity of 1 W/cm, amplitude of 40-60 μm, a wavelength 0,012 m, developed in Azerbaijan. Clinical and morphological methods, including examination of gingival tissue biopsies from the area where low-frequency ultrasound was applied, were used. After clinical and radiographic examination and identifying the impacted tooth with a favorable location on dental panoramic radiography, if there was a space in the tooth row, the area of the tooth projection at the gum was directly affected by low-frequency ultrasound daily or every other day for 5-10 seconds. An average of 1-5 procedures up to the tooth eruption was performed. Results. Microscopy of epithelial cover and stroma proved the absence of inflammation, thickening of outermost layer on the gum surface. Cells of the intermediate layer revealed the intact nucleus, loosening with linear swelling and cell dissociation. Cellular and tissue changes in gingival tissue seen after the treatment with low-frequency ultrasound may indicate the boosting of the impacted teeth eruption due to the syndrome of molecular contusion, improving the microcirculation and loosening the gum tissues. Conclusion. The results of comprehensive orthodontic treatment using low-frequency ultrasound ito stimulate the eruption of impacted teeth may be recommended for practical use.

Ultrastructure of Sheep Primordial Follicles Cultured in the Presence of Indol Acetic Acid, EGF, and FSH

The aim of this study was to investigate the ultrastructural characteristics of primordial follicles after culturing of sheep ovarian cortical slices in the presence of indol acetic acid (IAA), Epidermal Growth Factor (EGF), and FSH. To evaluate ultrastructure of primordial follicles cultured in MEM (control) or in MEM containing IAA, EGF, and FSH, fragments of cultured tissue were processes for transmission electron microscopy. Except in the control, primordial follicles cultured in supplemented media for 6 d were ultrastructurally normal. They had oocyte with intact nucleus and the cytoplasm contained heterogeneous-sized lipid droplets and numerous round or elongated mitochondria with intact parallel cristae were observed. Rough endoplasmic reticulum (RER) was rarely found. The granulosa cells cytoplasm contained a great number of mitochondria and abundant RER. In conclusion, the presence of IAA, EGF, and FSH helped to maintain ultrastructural integrity of sheep primordial follicles cultured in vitro.