Isolation and Culture of Larval Cells from C. elegans

Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×104 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Normative Values of Retinal Oxygen Saturation in Rhesus Monkeys: The Beijing Intracranial and Intraocular Pressure (iCOP) Study.

To study the normal values of the retinal oxygen saturation in Rhesus monkeys and to evaluated repeatability and reproducibility of retinal oxygen saturation measurements.Eighteen adult Rhesus macaque monkeys were included in this experimental study. An Oxymap T1 retinal oximeter (Oxymap, Reykjavik, Iceland) was used to perform oximetry on all subjects. Global arterial (SaO2) and venous oxygen saturation (SvO2), arteriovenous difference in SO2 were measured. In the first examination, each eye was imaged three times. At the following two examinations, each eye was imaged once. All examinations were finished in one month. P values were calculated to evaluate the difference between the measurements during three visits by performing an ANOVA. Intra-visit and inter-visit intraclass correlation coefficient (ICC) was determined.At baseline, the average SaO2 and SvO2 were 89.48 ± 2.64% and 54.85 ± 2.18%, respectively. The global A-V difference was 34.63 ± 1.91%. The difference between the three visits was not significant (p>0.05). The highest A-V difference in SO2 and lowest saturations were found in the inferotemporal quadrant. Intra-session and inter-visit repeatability were both high. For all oxygen saturation parameters, the ICC values of the intra-session repeatability ranged between 0.92 and 0.96. As found previously, a relatively high ICC value for inter-visit repeatability also was found for all oxygen saturation measurements, ranging between 0.86 and 0.94, with the lowest values in the infero-nasal quadrant.Our study is the first to describe retinal SO2 in healthy Rhesus monkeys. In normal monkey eyes, the reproducibility and repeatability of retinal oximetry oxygen saturation measurements were high in the retinal arterioles and venules. Our results support that Oxymap T1 retinal oximetry is a suitable and reliable technique in monkey studies

Evaluation of the relationship between maxillary posterior teeth and the maxillary sinus floor using cone-beam computed tomography

Abstract Background Maxillary posterior teeth have close anatomical proximity to the maxillary sinus floor (MSF), and the race, gender, age, side and presence/absence of adjacent teeth may influence the mean distances between the root apices and the MSF. This study aimed to evaluate both the relationship between the maxillary posterior teeth and MSF, and the influence of adjacent teeth loss on the distance between the maxillary posterior roots and MSF. Methods Cone-beam computed tomography images were collected from 1011 Chinese patients. The relationship between the maxillary posterior teeth and the MSF was divided into three types: Type OS (the root apex extending below/outside the MSF), Type CO (the root apex contacting with the MSF), Type IS (the root apex extending above/inside the MSF). The minimum vertical distances between the maxillary posterior roots apices and the MSF were recorded. The correlations of the distances with gender and age were analyzed. The distances between the maxillary posterior root apices and the MSF with different types of adjacent teeth loss was evaluated. Results Type OS was the most common relationship of all posterior root apices (P<0.05). Type IS was highest in the palatal roots (PRs) of the maxillary first molars (MFMs) and the mesiobuccal roots (MBRs) of the maxillary second molars (MSMs) (24.8% and 21.6%) (P<0.05). The frequency of Type IS decreased with age except the premolar roots and PRs of the MSMs (P<0.05). The MBRs of the MSMs had the lowest distances to the MSF (0.8 ± 2.5 mm), followed by the distobuccal roots of the MSMs (1.3 ± 2.7 mm) and the PRs of the MFMs (1.4 ± 3.4 mm) (P<0.05). Age was an important influencing factor to the mean distances while gender had little effects. The distance between the maxillary second premolar root apices and the MSF decreased with the absence of adjacent teeth (P<0.05). Conclusions The maxillary molars showed greater proximity to the MSF than premolars. Age had significant impacts on the relationship between maxillary posterior roots and MSF. The absence of maxillary first molars will influence the proximity of maxillary second premolar root apices to MSF

The effect of lateral decubitus position on nocturnal intraocular pressure over a habitual 24-hour period in healthy adults.

PURPOSE: To investigate the effect of lateral decubitus position (LDP) on nocturnal intraocular pressure (IOP) and the effect of LDP on 24-hour habitual IOP pattern in healthy subjects. METHODS: Intraocular pressure was measured every 2-hours using an Accupen Applanation Tonometer (Accutome, USA). During the diurnal period (7:30 am, 9:30 am, 11:30 am, 1:30 pm, 3:30 pm, 5:30 pm, 7:30 pm, and 9:30 pm), IOP was measured in the sitting position under bright light (500-1000 lux) after the subjects had been seated for 5 min. The nocturnal IOP was measured in the supine position, right LDP, and left LDP, with randomized sequences, under dim light (<10 lux) at 11:30 pm, 1:30 am, 3:30 am, and 5:30 am. The subjects were awakened and maintained each position for 5 min before the measurement. The 24-hour habitual IOP patterns were obtained according to the nocturnal position (supine, right LDP and left LDP) for either eye. P<0.05 was considered to be significant. RESULTS: Nineteen healthy subjects were included with a mean age of 51.3±5.8 years. During the nocturnal period, a significant IOP difference was found between the dependent eye (the eye on the lower side) of LDP and the supine position, but not for all the nocturnal time points. Over a 24-hour period, the effect of LDP on habitual IOP pattern was not statistically significant, although the mean nocturnal IOP and the diurnal-nocturnal IOP change for the right and the left eye in the LDP pattern was slightly higher than that in the sitting-supine pattern. CONCLUSION: Significant nocturnal IOP differences existed between the dependent eye and the supine, but did not occur consistently for all time points. Over a 24-hour period, the effect of LDP on habitual IOP pattern was not statistically significant in healthy subjects

Generation of Large-Scale Moderate-Resolution Forest Height Mosaic With Spaceborne Repeat-Pass SAR Interferometry and Lidar

This paper provides an overview of the scattering model, inversion approach, and validation of the application results for creating large-scale moderate-resolution (hectare-level) mosaics of forest height through using spaceborne repeat-pass SAR interferometry and lidar. By incorporating several improvements to the forest height inversion and mosaicking approach, the height estimation accuracy along with the robustness of this approach have been considerably enhanced from its originally reported accuracy of RMSE of 3–4 m at a 20-hectare aggregated pixel size to RMSE of 3–4 m on the order of 3–6 hectares. Furthermore, practical data processing schemes are provided in detail. Extensive validation results are demonstrated which include: 1) a forest height mosaic (total area of 11.6 million hectares) is generated for the U.S. states of Maine and New Hampshire using Japanese Aerospace Exploration Agency’s (JAXA) ALOS-1 InSAR correlation data and a small airborne lidar strip (44 000 hectares); 2) the mosaic height estimates are further compared with the available airborne lidar data and field measurements over both flat and mountainous areas; and 3) feasibility of using modern repeat-pass InSAR satellites with short repeat interval is also examined by using JAXA’s ALOS-2 data. This simple and efficient approach is a potential observational prototype with much smaller error budget for the future spaceborne repeat-pass L-band InSAR systems with small spatial baseline and moderate/large temporal baseline (such as NISAR) in combination with lidar (such as GEDI) on the application of large-scale forest height/biomass mapping. It also serves as a complementary tool to the spaceborne single-pass InSAR systems using InSAR/PolInSAR methods when full-pol data are not available and/or when the underlying topography slope causes problems for these approaches