Hemoglobin Absorption Spectral Imaging (H.A.S.I.): a novel optical staining technique for microlaryngoscopy.

PURPOSE: Optical image enhancement techniques are widely used in endoscopy to improve the visualization of blood vessels for diagnostic and therapeutic purposes. These techniques are monitor-based and therefore not available for direct microscopy. In this study, a novel optical microscope filter, Hemoglobin absorption spectral imaging (H.A.S.I.) was tested for use in microlaryngoscopy. METHODS: A novel dichroic filter was designed to improve contrast in small blood vessels by highlighting transmission in the spectrum range of hemoglobin absorption maxima. A surgical microscope equipped with the novel H.A.S.I. filter was installed in one operating room in our institution. 68 consecutive patients referred to our ENT department for endoscopy were examined using white light and the novel H.A.S.I. filter during microlaryngoscopy. Surgeons described the blood vessels of the vocal cords using a classification chart and assessed for suspected malignancy using both white light and H.A.S.I. RESULTS: 77 consecutive microlaryngoscopies were performed on 68 patients. 142 vocal cords were visualized in microlaryngoscopy and the blood vessels classified according to the chart. With white light, 152 blood vessel characteristics were documented and 157 with H.A.S.I. Notably, pathologies like benign horizontal blood vessel changes, leukoplakia, and vertical blood vessel changes like dots and loops were seen more frequently with H.A.S.I. Finally, seven lesions were treated by transoral laser microsurgery (TLM) with H.A.S.I. to test the practicability of the method for microlaryngoscopic laser surgery. CONCLUSION: This is the first study describing H.A.S.I. as an optical staining method for microlaryngoscopy. In our experience, the method was practical and improved the evaluation of vocal cord blood vessels. In some cases, the use of H.A.S.I. led to a change in diagnosis and treatment. Also, H.A.S.I. was found to be helpful in microlaryngeal laser surgery for demarcating resection margins. This is, to our knowledge, the first optical staining method integrated into a surgical microscope and can be conveniently used during microlaryngeal laser surgery and does not require further equipment.

Protein Tpr is required for establishing nuclear pore-associated zones of heterochromatin exclusion.

Amassments of heterochromatin in somatic cells occur in close contact with the nuclear envelope (NE) but are gapped by channel- and cone-like zones that appear largely free of heterochromatin and associated with the nuclear pore complexes (NPCs). To identify proteins involved in forming such heterochromatin exclusion zones (HEZs), we used a cell culture model in which chromatin condensation induced by poliovirus (PV) infection revealed HEZs resembling those in normal tissue cells. HEZ occurrence depended on the NPC-associated protein Tpr and its large coiled coil-forming domain. RNAi-mediated loss of Tpr allowed condensing chromatin to occur all along the NE's nuclear surface, resulting in HEZs no longer being established and NPCs covered by heterochromatin. These results assign a central function to Tpr as a determinant of perinuclear organization, with a direct role in forming a morphologically distinct nuclear sub-compartment and delimiting heterochromatin distribution.

TOR signaling regulates liquid phase separation of the SMN complex governing snRNP biogenesis.

The activity of the SMN complex in promoting the assembly of pre-mRNA processing UsnRNPs correlates with condensation of the complex in nuclear Cajal bodies. While mechanistic details of its activity have been elucidated, the molecular basis for condensation remains unclear. High SMN complex phosphorylation suggests extensive regulation. Here, we report on systematic siRNA-based screening for modulators of the capacity of SMN to condense in Cajal bodies and identify mTOR and ribosomal protein S6 kinase ß-1 as key regulators. Proteomic analysis reveals TOR-dependent phosphorylations in SMN complex subunits. Using stably expressed or optogenetically controlled phospho mutants, we demonstrate that serine 49 and 63 phosphorylation of human SMN controls the capacity of the complex to condense in Cajal bodies via liquid-liquid phase separation. Our findings link SMN complex condensation and UsnRNP biogenesis to cellular energy levels and suggest modulation of TOR signaling as a rational concept for therapy of the SMN-linked neuromuscular disorder spinal muscular atrophy.

δ18O in the tropical conifer Agathis robusta records ENSO-related precipitation variations.

Long-lived trees from tropical Australasia are a potential source of information about internal variability of the El Niño-Southern Oscillation (ENSO), because they occur in a region where precipitation variability is closely associated with ENSO activity. We measured tree-ring width and oxygen isotopic composition (δ18O) of α-cellulose from Agathis robusta (Queensland Kauri) samples collected in the Atherton Tablelands, Queensland, Australia. Standard ring-width chronologies yielded low internal consistency due to the frequent presence of false ring-like anatomical features. However, in a detailed examination of the most recent 15 years of growth (1995-2010), we found significant correlation between δ18O and local precipitation, the latter associated with ENSO activity. The results are consistent with process-based forward modeling of the oxygen isotopic composition of α-cellulose. The δ18O record also enabled us to confirm the presence of a false growth ring in one of the three samples in the composite record, and to determine that it occurred as a consequence of anomalously low rainfall in the middle of the 2004/5 rainy season. The combination of incremental growth and isotopic measures may be a powerful approach to development of long-term (150+ year) ENSO reconstructions from the terrestrial tropics of Australasia.

On the influence of the architecture of poly(ethylene glycol)-based thermoresponsive polymers on cell adhesion.

Thermoresponsive polymer surface coatings are a promising tool for cell culture applications. They allow for a mild way of cell detachment that preserves the activity of membrane proteins-a prerequisite for reliable cell analysis. To enlarge the application range of these coatings to cells with different adhesion properties, we synthesized various novel poly(ethylene glycol)-based thermoresponsive polymers and describe how (i) their chemical structure and (ii) their surface density affect their efficiency. In order to quantify the influence of both factors, the time for cell spreading and rounding efficiency were observed. As a result, efficiency of cell rounding, which is closely correlated to cell detachment, is less affected by both factors than the time needed for cell spreading. This time can effectively be adjusted by the molecular architecture which includes the length of the polymer backbone and the side chains. Based on this work, recommendations are given for future optimization of functionality of thermoresponsive polymer coatings for cell culture applications.