Parallelized Droplet Vitrification Enables Single-Run Vitrification of the Whole Rat Liver Hepatocyte Yield.

Drug discovery pipelines rely on the availability of isolated primary hepatocytes for investigating potential hepatotoxicity prior to clinical application. These hepatocytes are typically isolated from livers rejected for transplantation and subsequently cryopreserved for later usage. The gold-standard cryopreservation technique, slow-freezing, is a labor-intensive process, with significant post-storage viability loss. In this work, we introduce parallelized droplet vitrification, a technique for high-volumetric, rapid vitrification of suspended cells. We show the utility of this technique through the single-run vitrification of the whole-rate liver hepatocyte yield, resulting in a 1600% increase in single-batch vitrification and a 500% increase in droplet generation rate compared to previous droplet vitrification approaches. Additionally, we showed that these implementations maintained improved post-preservation outcomes in primary rat hepatocytes.

Robotic rectal cancer surgery: comparative study of the impact of obesity on early outcomes.

The aim of this study was to compare the outcomes of robotic total mesorectal excision (TME) in obese versus non-obese patients. A total of 533 patients, of whom 161 were obese (30·2 per cent) underwent robotic proctectomy during the study interval. Patient obesity was not associated with adverse short-term clinical outcomes after robotic rectal cancer surgery. Indicated in the obese perhaps?

Robotic lateral pelvic lymph node dissection after chemoradiation for rectal cancer: a Western perspective.

AIM: There are limited outcome data for lateral pelvic lymph node dissection (LPLND) following neoadjuvant chemoradiotherapy (nCRT), particularly in the West. Our aim was to evaluate the short-term perioperative and oncological outcomes of robotic LPLND at a single cancer centre. METHOD: A retrospective analysis of a prospective database of consecutive patients undergoing robotic LPLND for rectal cancer between November 2012 and February 2020 was performed. The main outcomes were short-term perioperative and oncological outcomes. Major morbidity was defined as Clavien-Dindo grade 3 or above. RESULTS: Forty patients underwent robotic LPLND during the study period. The mean age was 54 years (SD ± 15 years) and 13 (31.0%) were female. The median body mass index was 28.6 kg/m2 (IQR 25.5-32.6 kg/m2 ). Neoadjuvant CRT was performed in all patients. Resection of the primary rectal cancer and concurrent LPLND occurred in 36 (90.0%) patients, whilst the remaining 4 (10.0%) patients had subsequent LPLND after prior rectal resection. The median operating time was 420 min (IQR 313-540 min), estimated blood loss was 150 ml (IQR 55-200 ml) and length of hospital stay was 4 days (IQR 3-6 days). The major morbidity rate was 10.0% (n = 4). The median lymph node harvest from the LPLND was 6 (IQR 3-9) and 13 (32.5%) patients had one or more positive LPLNs. The median follow-up was 16 months (IQR 5-33 months), with 1 (2.5%) local central recurrence and 7 (17.5%) patients developing distant disease, resulting in 3 (7.5%) deaths. CONCLUSION: Robotic LPLND for rectal cancer can be performed in Western patients to completely resect extra-mesorectal LPLNs and is associated with acceptable perioperative morbidity.

Retrofitting an environmental monitor with a silicon photomultiplier sensor.

We report on the retrofitting of a standard DP2 environmental radiation monitor replacing the photomultiplier tube with a silicon photomultiplier (SiPM). The use of a SiPM has several advantages for a hand-held radiation monitor, including convenient low-voltage operation and physical robustness. We report the detection efficiency and alpha/beta discrimination performance of the modified probe compared with an unmodified version.

Selective central vascular ligation (D3 lymphadenectomy) in patients undergoing minimally invasive complete mesocolic excision for colon cancer: optimizing the risk-benefit equation.

AIM: Complete mesocolic excision (CME) with central vascular ligation (CVL) has been advocated for right colon adenocarcinoma (RC), but the radicality of vascular dissection remains controversial. Our aim is to report outcomes of selective CVL (D3 lymphadenectomy) during minimally invasive CME for RC. METHOD: A prospective database identified patients who were treated for RC between 2009 and 2016. Minimally invasive CME was standard. The radicality of lymphadenectomy was defined as high ligation (HL) versus CVL based on operative reports and videos. Two blinded radiologists independently evaluated the pre- and postoperative CT scans for radiographically abnormal nodes. RESULTS: Of 197 patients who underwent CME, HL was performed in 56 (28%) and CVL in 141 (72%). There were no baseline differences in age, sex, body mass index, American Society of Anesthesiologists score or pathological staging, and there were no major intra-operative complications in either group (including no major vascular injuries). The median total number of nodes retrieved was 27 and 31 (P = 0.011) in HL and CVL groups, resepctively, with pathologically positive nodes identified in 33.9% and 39.8% (P = 0.704), respectively. Preoperative imaging identified abnormal cN3 nodes in 1.5% of patients; all of whom underwent CVL. No abnormal cN2 or cN3 nodes remained on postoperative imaging. The 60-day mortality was 0.5%, and major morbidity was 4%. One patient (0.5%) had an anastomotic recurrence after a median follow-up of 22 months. CONCLUSION: With imperfect preoperative clinical nodal staging, and in the absence of randomized data, the low morbidity and oncological outcomes observed support the approach of CME with HL as a minimum standard, with CVL (D3 lymphadenectomy) in selected cases.

Evaluation of Scintillator Detection Materials for Application within Airborne Environmental Radiation Monitoring.

In response to the Fukushima Daiichi Nuclear Power Plant accident, there has occurred the unabated growth in the number of airborne platforms developed to perform radiation mapping-each utilising various designs of a low-altitude uncrewed aerial vehicle. Alongside the associated advancements in the airborne system transporting the radiation detection payload, from the earliest radiological analyses performed using gas-filled Geiger-Muller tube detectors, modern radiation detection and mapping platforms are now based near-exclusively on solid-state scintillator detectors. With numerous varieties of such light-emitting crystalline materials now in existence, this combined desk and computational modelling study sought to evaluate the best-available detector material compatible with the requirements for low-altitude autonomous radiation detection, localisation and subsequent high spatial-resolution mapping of both naturally occurring and anthropogenically-derived radionuclides. The ideal geometry of such detector materials is also evaluated. While NaI and CsI (both elementally doped) are (and will likely remain) the mainstays of radiation detection, LaBr3 scintillation detectors were determined to possess not only a greater sensitivity to incident gamma-ray radiation, but also a far superior spectral (energy) resolution over existing and other potentially deployable detector materials. Combined with their current competitive cost, an array of three such composition cylindrical detectors were determined to provide the best means of detecting and discriminating the various incident gamma-rays.

Colorectal premalignancy is associated with consensus molecular subtypes 1 and 2.

Background: Gene expression-based profiling of colorectal cancer (CRC) can be used to identify four molecularly homogeneous consensus molecular subtype (CMS) groups with unique biologic features. However, its applicability to colorectal premalignant lesions remains unknown. Patients and methods: We assembled the largest transcriptomic premalignancy dataset by integrating different public and proprietary cohorts of adenomatous and serrated polyps from sporadic (N = 311) and hereditary (N = 78) patient populations and carried out a comprehensive analysis of carcinogenesis pathways using the CMS random forest (RF) classifier. Results: Overall, transcriptomic subtyping of sporadic and hereditary polyps revealed CMS2 and CMS1 subgroups as the predominant molecular subtypes in premalignancy. Pathway enrichment analysis showed that adenomatous polyps from sporadic or hereditary cases (including Lynch syndrome) displayed a CMS2-like phenotype with WNT and MYC activation, whereas hyperplastic and serrated polyps with CMS1-like phenotype harbored prominent immune activation. Rare adenomas with CMS4-like phenotype showed significant enrichment for stromal signatures along with transforming growth factor-ß activation. There was a strong association of CMS1-like polyps with serrated pathology, right-sided anatomic location and BRAF mutations. Conclusions: Based on our observations made in premalignancy, we propose a model of pathway activation associated with CMS classification in colorectal carcinogenesis. Specifically, while adenomatous polyps are largely CMS2, most hyperplastic and serrated polyps are CMS1 and may transition into other CMS groups during evolution into carcinomas. Our findings shed light on the transcriptional landscape of premalignant colonic polyps and may help guide the development of future biomarkers or preventive treatments for CRC.

A guide to analysis and reconstruction of serial block face scanning electron microscopy data.

Serial block face scanning electron microscopy (SBF-SEM) is a relatively new technique that allows the acquisition of serially sectioned, imaged and digitally aligned ultrastructural data. There is a wealth of information that can be obtained from the resulting image stacks but this presents a new challenge for researchers - how to computationally analyse and make best use of the large datasets produced. One approach is to reconstruct structures and features of interest in 3D. However, the software programmes can appear overwhelming, time-consuming and not intuitive for those new to image analysis. There are a limited number of published articles that provide sufficient detail on how to do this type of reconstruction. Therefore, the aim of this paper is to provide a detailed step-by-step protocol, accompanied by tutorial videos, for several types of analysis programmes that can be used on raw SBF-SEM data, although there are more options available than can be covered here. To showcase the programmes, datasets of skeletal muscle from foetal and adult guinea pigs are initially used with procedures subsequently applied to guinea pig cardiac tissue and locust brain. The tissue is processed using the heavy metal protocol developed specifically for SBF-SEM. Trimmed resin blocks are placed into a Zeiss Sigma SEM incorporating the Gatan 3View and the resulting image stacks are analysed in three different programmes, Fiji, Amira and MIB, using a range of tools available for segmentation. The results from the image analysis comparison show that the analysis tools are often more suited to a particular type of structure. For example, larger structures, such as nuclei and cells, can be segmented using interpolation, which speeds up analysis; single contrast structures, such as the nucleolus, can be segmented using the contrast-based thresholding tools. Knowing the nature of the tissue and its specific structures (complexity, contrast, if there are distinct membranes, size) will help to determine the best method for reconstruction and thus maximize informative output from valuable tissue.

The use of toxicokinetics and exposure studies to show that carprofen in cattle tissue could lead to secondary toxicity and death in wild vultures.

Veterinary medicines can be extremely damaging to the environment, as seen with the catastrophic declines in Gyps vulture in South Asia due to their secondary exposure to diclofenac in their primary food source. Not surprisingly, concern has been raised over other similar drugs. In this study, we evaluate the toxicity of carprofen to the Gyps vulture clade through plasma pharmacokinetics evaluations in Bos taurus cattle (their food source) and Gyps africanus (a validated model species); tissue residues in cattle; and the effect of carprofen as a secondary toxicant as both tissue-bound residue or pure drug at levels expected in cattle tissues. Carprofen residues were highest in cattle kidney (7.72 ± 2.38 mg/kg) and injection site muscle (289.05 ± 98.96 mg/kg of dimension of 5 × 5 × 5 cm). Vultures exposed to carprofen as residues in the kidney tissue or pure drug equivalents showed no toxic signs. When exposed to average injection site concentrations (64 mg/kg) one of two birds died with evidence of severe renal and liver damage. Toxicokinetic analysis revealed a prolonged drug half-life of 37.75 h in the dead bird as opposed to 13.99 ± 5.61 h from healthy birds dosed intravenously at 5 mg/kg. While carprofen may generally be harmless to Gyps vultures, its high levels at the injection site in treated cattle can result in lethal exposure in foraging vultures, due to relative small area of tissue it is found therein. We thus suggest that carprofen not be used in domesticated ungulates in areas where carcasses are accessible or provided to vultures at supplementary feeding sites.

Metabolism of aceclofenac in cattle to vulture-killing diclofenac.

The nonsteroidal anti-inflammatory drug (NSAID) diclofenac is highly toxic to Gyps vultures, and its recent widespread use in South Asia caused catastrophic declines in at least 3 scavenging raptors. The manufacture of veterinary formulations of diclofenac has since been banned across the region with mixed success. However, at least 12 other NSAIDs are available for veterinary use in South Asia. Aceclofenac is one of these compounds, and it is known to metabolize into diclofenac in some mammal species. The metabolic pathway of aceclofenac in cattle, the primary food of vultures in South Asia, is unknown. We gave 6 cattle the recommended dose of aceclofenac (2 mg/kg), collected blood thereafter at intervals for up to 12 h, and used liquid chromatography with mass spectrometry in a pharmacokinetic analysis of aceclofenac and diclofenac in the plasma. Nearly all the aceclofenac administered to the cattle was very rapidly metabolized into diclofenac. At 2 h, half the aceclofenac had been converted into diclofenac, and at 12 h four-fifths of the aceclofenac had been converted into diclofenac. Therefore, administering aceclofenac to livestock poses the same risk to vultures as administering diclofenac to livestock. This, coupled with the risk that aceclofenac may replace diclofenac in the veterinary market, points to the need for an immediate ban on all aceclofenac formulations that can be used to treat livestock. Without such a ban, the recovery of vultures across South Asia will not be successful.

Sunny with a chance of gastroenteritis: predicting swimmer risk at California beaches.

Traditional beach management that uses concentrations of cultivatable fecal indicator bacteria (FIB) may lead to delayed notification of unsafe swimming conditions. Predictive, nowcast models of beach water quality may help reduce beach management errors and enhance protection of public health. This study compares performances of five different types of statistical, data-driven predictive models: multiple linear regression model, binary logistic regression model, partial least-squares regression model, artificial neural network, and classification tree, in predicting advisories due to FIB contamination at 25 beaches along the California coastline. Classification tree and the binary logistic regression model with threshold tuning are consistently the best performing model types for California beaches. Beaches with good performing models usually have a rainfall/flow related dominating factor affecting beach water quality, while beaches having a deteriorating water quality trend or low FIB exceedance rates are less likely to have a good performing model. This study identifies circumstances when predictive models are the most effective, and suggests that using predictive models for public notification of unsafe swimming conditions may improve public health protection at California beaches relative to current practices.

Predicting water quality at Santa Monica Beach: evaluation of five different models for public notification of unsafe swimming conditions.

Bathing beaches are monitored for fecal indicator bacteria (FIB) to protect swimmers from unsafe conditions. However, FIB assays take ∼24 h and water quality conditions can change dramatically in that time, so unsafe conditions cannot presently be identified in a timely manner. Statistical, data-driven predictive models use information on environmental conditions (i.e., rainfall, turbidity) to provide nowcasts of FIB concentrations. Their ability to predict real time FIB concentrations can make them more accurate at identifying unsafe conditions than the current method of using day or older FIB measurements. Predictive models are used in the Great Lakes, Hong Kong, and Scotland for beach management, but they are presently not used in California - the location of some of the world's most popular beaches. California beaches are unique as point source pollution has generally been mitigated, the summer bathing season receives little to no rainfall, and in situ measurements of turbidity and salinity are not readily available. These characteristics may make modeling FIB difficult, as many current FIB models rely heavily on rainfall or salinity. The current study investigates the potential for FIB models to predict water quality at a quintessential California Beach: Santa Monica Beach. This study compares the performance of five predictive models, multiple linear regression model, binary logistic regression model, partial least square regression model, artificial neural network, and classification tree, to predict concentrations of summertime fecal coliform and enterococci concentrations. Past measurements of bacterial concentration, storm drain condition, and tide level are found to be critical factors in the predictive models. The models perform better than the current beach management method. The classification tree models perform the best; for example they correctly predict 42% of beach postings due to fecal coliform exceedances during model validation, as compared to 28% by the current method. Artificial neural network is the second best model which minimizes the number of incorrect beach postings. The binary logistic regression model also gives promising results, comparable to classification tree, by adjusting the posting decision thresholds to maximize correct beach postings. This study indicates that predictive models hold promise as a beach management tool at Santa Monica Beach. However, there are opportunities to further refine predictive models.

Urinary excretion of arsenic following rice consumption.

Patterns of arsenic excretion were followed in a cohort (n = 6) eating a defined rice diet, 300 g per day d.wt. where arsenic speciation was characterized in cooked rice, following a period of abstinence from rice, and other high arsenic containing foods. A control group who did not consume rice were also monitored. The rice consumed in the study contained inorganic arsenic and dimethylarsinic acid (DMA) at a ratio of 1:1, yet the urine speciation was dominated by DMA (90%). At steady state (rice consumption/urinary excretion) ∼40% of rice derived arsenic was excreted via urine. By monitoring of each urine pass throughout the day it was observed that there was considerable variation (up to 13-fold) for an individual's total arsenic urine content, and that there was a time dependent variation in urinary total arsenic content. This calls into question the robustness of routinely used first pass/spot check urine sampling for arsenic analysis.

Persistence of brodifacoum in cockroach and woodlice: implications for secondary poisoning during rodent eradications.

When planning rodent eradications, that normally involve the use of the anticoagulant poison brodifacoum, it is imperative to minimise impacts on other "non-target" species that dwell alongside the targeted rodents and may indeed be the intended beneficiaries of the eradication. Such impacts can arise either from primary poisoning when the non-target species ingest bait pellets containing toxicant or by secondary poisoning when the non-target species eats prey that has itself eaten brodifacoum. Cockroaches and woodlice, likely to scavenge bait pellets, are widely distributed on tropical and sub-tropical islands where they are eaten by ground-dwelling birds. Combining work on Henderson Island, South Pacific, site of a recent rat eradication project, and UK laboratory experiments, our study first measured brodifacoum concentrations in cockroaches given temporary ad lib access to poison bait pellets, approximately mimicking the aftermath of bait distribution for a rodent eradication. In two separate experiments using different species/exposure times, the mean brodifacoum concentrations among cockroaches immediately after bait exposure was 262±s.e. 131 and 477±168µgkg(-1) wet weight. Values decreased quickly in the following 2 weeks, and then continued to decline at a slower rate over the following 4 weeks in the more prolonged laboratory experiment. A supplementary experiment with woodlice recorded a similar brodifacoum concentration in the animals at the end of the exposure period, 223±66µgkg(-1), and a similar time course for the post-exposure decline. In the context of rails (Rallidae), a group of birds known to be particularly susceptible to primary brodifacoum poisoning, these results suggested that, in terms of acute exposure, individual birds would need to eat a minimum of their own body weight (and more commonly 2-5 times that) of live cockroaches before facing a 50% risk of death. Therefore, we conclude that in eradication scenarios, acute secondary poisoning is of lower concern for these birds than primary poisoning.

Differential vasodilation of human placental and myometrial arteries related to myofilament Ca(2+)-desensitization and the expression of Hsp20 but not MYPT1.

Endothelial-dependent regulation of vascular tone occurs in part via protein kinase G1α-mediated changes in smooth muscle myofilament sensitivity to Ca(2+). Tissue-specific differences in PKG-dependent relaxation have been attributed to altered expression of myofilament-associated proteins that are substrates for PKG binding. These include the alternative splicing of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase to yield leucine zipper positive (LZ(+)) and negative (LZ(-)) isovariants, with the former being required for PKG-mediated relaxation, and/or altered expressions of telokin, vasodilator-stimulated phosphoprotein (VASP) or heat shock protein Hsp20. During human pregnancy the uterine and placental circulations remain distinct entities and, as such, their mechanisms of vascular tone regulation may differ. Indeed, the sensitivity of myometrial arteries to endothelial-dependent agonists has been suggested to be greater than that of placental arteries. We tested the hypothesis that this was related to tissue-specific changes in PKG-mediated myofilament Ca(2+)-desensitization and/or the expressions of PKG-interacting myofilament-associated proteins. Permeabilized human placental and myometrial arteries were constricted with maximal activating Ca(2+) (pCa 4.5), or sub-maximal Ca(2+) (pCa 6.7) and the thrombane mimetic U46619, and exposed to 8-Br-cGMP. In each case, relaxation was significantly greater in myometrial arteries (e.g. relaxation in pCa 4.5 to 8-Br-cGMP was 49 ± 9.7%, n = 7) than placental arteries (relaxation of 23 ± 6.6%, n = 6, P < 0.05). MYPT1 protein levels, or MYPT1 LZ(+)/LZ(-) mRNA ratios, were similar for both artery types. Of other proteins examined, only Hsp20 expression was significantly elevated in myometrial arteries than placental arteries. These results demonstrate that the reduced human placental artery relaxation to PKG stimulation lies partly at the level of myofilament (de)activation and may be related to a lower expression of Hsp20 than in myometrial arteries.

Isolated small uterine arteries from non-pregnant and term pregnant rats exhibit regulatory responses to elevations in extravascular pressure.

An adequate blood supply is essential for the maintenance of uterine function and fetal health during parturition. However, labouring uterine contractions will impart compressive forces on small uterine arteries (SUA). We demonstrate that isolated, pressurised rat SUA arteries, pre-constricted with arginine vasopressin or high potassium solution, exhibit regulatory responses to elevations in extravascular pressure (EVP) which maintain internal diameter constant at EVPs of 0-40 mm Hg. This response is endothelium independent and is not modulated by pregnancy. No regulation was observed in calcium free solution. SUA myogenic responses to elevated EVP likely represents a mechanism for limiting reductions in uterine blood flow during uterine contraction.

Measurement of the 18Ne(α,p0) 21Na reaction cross section in the burning energy region for x-ray bursts.

The 18Ne(α,p) 21Na reaction provides one of the main HCNO-breakout routes into the rp process in x-ray bursts. The 18Ne(α,p0) 21Na reaction cross section has been determined for the first time in the Gamow energy region for peak temperatures T∼2 GK by measuring its time-reversal reaction 21Na(p,α) 18Ne in inverse kinematics. The astrophysical rate for ground-state to ground-state transitions was found to be a factor of 2 lower than Hauser-Feshbach theoretical predictions. Our reduced rate will affect the physical conditions under which breakout from the HCNO cycles occurs via the 18Ne(α,p) 21Na reaction.

Systemic chemotherapy and surgical cytoreduction for poorly differentiated and signet ring cell adenocarcinomas of the appendix.

BACKGROUND: Poorly differentiated and signet ring cell adenocarcinomas of the appendix represent a subset with aggressive tumor biology and poor outcomes with few studies evaluating the impact of systemic chemotherapy and cytoreductive surgery (CRS). PATIENTS AND METHODS: A retrospective chart review of patients with either poorly differentiated and signet ring cell appendiceal adenocarcinomas was completed from 1992 to 2010. RESULTS: One hundred forty-two patients were identified. Seventy-eight patients with metastatic disease received chemotherapy. Radiographic response was 44%, median progression-free survival (PFS) was 6.9 months, and median overall survival (OS) was 1.7 years. In multivariate analysis, response to chemotherapy [hazard ratio (HR) 0.5; P = 0.02] predicted improved PFS, and complete CRS (HR 0.3; P = 0.004) predicted improved OS. Patients who underwent complete CRS (n = 26) had a median relapse-free survival (RFS) of 1.2 years and a median OS of 4.2 years. In multivariate analysis for this subset, complete cytoreduction score of 0 was significantly correlated with improved RFS (HR 0.07; P = 0.01) and OS (HR 0.02; P = 0.01). CONCLUSIONS: Systemic chemotherapy appears to be a viable treatment option for patients with metastatic poorly differentiated and signet ring cell appendiceal adenocarcinomas. Complete CRS is associated with improved RFS and OS, though part of this benefit likely reflects the selection of good tumor biology.

Towards a computational reconstruction of the electrodynamics of premature and full term human labour.

We apply virtual tissue engineering to the full term human uterus with a view to reconstruction of the spatiotemporal patterns of electrical activity of the myometrium that control mechanical activity via intracellular calcium. The three-dimensional geometry of the gravid uterus has been reconstructed from segmented in vivo magnetic resonance imaging as well as ex vivo diffusion tensor magnetic resonance imaging to resolve fine scale tissue architecture. A late-pregnancy uterine smooth muscle cell model is constructed and bursting analysed using continuation algorithms. These cell models are incorporated into partial differential equation models for tissue synchronisation and propagation. The ultimate objective is to develop a quantitative and predictive understanding of the mechanisms that initiate and regulate labour.