-
Seminars in Thrombosis and Hemostasis Feb 2024There is no certainty in change, other than change is certain. As celebrates 50 years of publication, I felt it appropriate to reflect on my own 40-year plus scientific...
There is no certainty in change, other than change is certain. As celebrates 50 years of publication, I felt it appropriate to reflect on my own 40-year plus scientific career. My career in the thrombosis and hemostasis field did not start until 1987, but the subsequent 35 years reflected a period of significant change in associated disease diagnostics. I started in the Westmead Hospital "coagulation laboratory" when staff were still performing manual clotting tests, using stopwatches, pipettes, test tubes, and a water bath, which we transported to the hospital outpatient department to run our weekly warfarin clinic. Several hemostasis instruments have come and gone, including the Coag-A-Mate X2, the ACL-300R, the MDA-180, the BCS XP, and several StaR Evolution analyzers. Some instruments remain, including the PFA-100, PFA-200, the AggRAM, the CS-5100, an AcuStar, a Hydrasys gel system, and two ACL-TOP 750s. We still have a water bath, but this is primarily used to defrost frozen samples, and manual clotting tests are only used to teach visiting medical students. We have migrated across several methodologies in the 45-year history of the local laboratory. Laurel gel rockets, used for several assays in the 1980s, were replaced with enzyme-linked immunosorbent assay assays and most assays were eventually placed on automated instruments. Radio-isotopic assays, used in the 1980s, were replaced by an alternate safer method or else abandoned. Test numbers have increased markedly over time. The approximately 31,000 hemostasis assays performed at the Westmead-based laboratory in 1983 had become approximately 200,000 in 2022, a sixfold increase. Some 90,000 prothrombin times and activated partial thromboplastic times are now performed at this laboratory per year. Thrombophilia assays were added to the test repertoires over time, as were the tests to measure several anticoagulant drugs, most recently the direct oral anticoagulants. I hope my personal history, reflecting on the changes in hemostasis testing over my career to date in the field, is found to be of interest to the readership, and I hope they forgive any inaccuracies I have introduced in this reflection of the past.
Topics: Humans; Hemostasis; Blood Coagulation Tests; Blood Coagulation; Thrombosis; Water
PubMed: 36731486
DOI: 10.1055/s-0043-1761487 -
Experimental Neurobiology Feb 2024The benefit of intranasal brain derived neurotrophic factor (BDNF) treatment on cognitive function in a neonatal postnatal day 7 (P7) mouse model of hypoxic ischemia...
The benefit of intranasal brain derived neurotrophic factor (BDNF) treatment on cognitive function in a neonatal postnatal day 7 (P7) mouse model of hypoxic ischemia (HI) was explored. Intranasal delivery is attractive in that it can promote widespread distribution of BDNF within both the brain and spinal cord. In this study we evaluated the effectiveness of intranasal BDNF to improve cognitive recovery following HI. HI is induced via ligation of the right carotid artery followed by a 45-minute exposure to an 8% oxygen/ 92% nitrogen mixture in an enclosed chamber. Male and female pups were subjected to a 2-hour hypothermia in a temperature-controlled chamber as a standard of care. A solution of saline (control) or recombinant human BDNF (Harlan Laboratories) was administered with a Gilson pipette at the same time each day for 7 days into each nasal cavity in awake mice beginning 24 hours after HI. We evaluated cognitive recovery using the novel object recognition (NOR) and western analysis to analyze neuro-markers and brain health such as synaptophysin and microtubule associated protein -2 (MAP2). The objective of this study was to evaluate the role and therapeutic potential of BDNF in neonatal HI recovery. Our results indicate that intranasal BDNF delivered within 24 hours after HI improved object discrimination at both 28 and 42 days after HI. Our results also demonstrate increased synaptophysin and MAP2 at day 42 in HI animals that received intranasal BDNF treatment compared to HI animals that were administered saline.
PubMed: 38471802
DOI: 10.5607/en23030 -
Asian Journal of Andrology Feb 2024Ex vivo tissue culture of the human corpus cavernosum (CC) can be used to explore the tissue structural changes and complex signaling networks. At present, artificial...
Ex vivo tissue culture of the human corpus cavernosum (CC) can be used to explore the tissue structural changes and complex signaling networks. At present, artificial CC-like tissues based on acellular or three-dimensional (3D)-printed scaffolds are used to solve the scarcity of primary penis tissue samples. However, inconvenience and high costs limit the wide application of such methods. Here, we describe a simple, fast, and economical method of constructing artificial CC-like tissue. Human CC fibroblasts (FBs), endothelial cells (ECs), and smooth muscle cells (SMCs) were expanded in vitro and mixed with Matrigel in specific proportions. A large number of bubbles were formed in the mixture by vortexing combined with pipette blowing, creating a porous, spongy, and spatial structure. The CC FBs produced a variety of signaling factors, showed multidirectional differentiation potential, and grew in a 3D grid in Matrigel, which is necessary for CC-like tissue to maintain a porous structure as a cell scaffold. Within the CC-like tissue, ECs covered the surface of the lumen, and SMCs were located inside the trabeculae, similar to the structure of the primary CC. Various cell components remained stable for 3 days in vitro, but the EC content decreased on the 7th day. Wingless/integrated (WNT) signaling activation led to lumen atrophy and increased tissue fibrosis in CC-like tissue, inducing the same changes in characteristics as in the primary CC. This study describes a preparation method for human artificial CC-like tissue that may provide an improved experimental platform for exploring the function and structure of the CC and conducting drug screening for erectile dysfunction therapy.
PubMed: 38319194
DOI: 10.4103/aja202374 -
APL Bioengineering Jun 2024Micropipette aspiration (MPA) is one of the gold standards for quantifying biological samples' mechanical properties, which are crucial from the cell membrane scale to...
Micropipette aspiration (MPA) is one of the gold standards for quantifying biological samples' mechanical properties, which are crucial from the cell membrane scale to the multicellular tissue. However, relying on the manipulation of individual home-made glass pipettes, MPA suffers from low throughput and no automation. Here, we introduce the sliding insert micropipette aspiration method, which permits parallelization and automation, thanks to the insertion of tubular pipettes, obtained by photolithography, within microfluidic channels. We show its application both at the lipid bilayer level, by probing vesicles to measure membrane bending and stretching moduli, and at the tissue level by quantifying the viscoelasticity of 3D cell aggregates. This approach opens the way to high-throughput, quantitative mechanical testing of many types of biological samples, from vesicles and individual cells to cell aggregates and explants, under dynamic physico-chemical stimuli.
PubMed: 38894959
DOI: 10.1063/5.0193333 -
Sensors (Basel, Switzerland) Jan 2024In recent years, the demand for effective intracytoplasmic sperm injection (ICSI) for the treatment of male infertility has increased. The ICSI operation is complicated...
In recent years, the demand for effective intracytoplasmic sperm injection (ICSI) for the treatment of male infertility has increased. The ICSI operation is complicated as it involves delicate organs and requires a high level of skill. Several cell manipulation systems that do not require such skills have been proposed; notably, several automated methods are available for cell rotation. However, these methods are unfeasible for the delicate ICSI medical procedure because of safety issues. Thus, this study proposes a microscopic system that enables intuitive micropipette manipulation using a haptic device that safely and efficiently performs the entire ICSI procedure. The proposed system switches between field-of-view expansion and three-dimensional image presentation to present images according to the operational stage. In addition, the system enables intuitive pipette manipulation using a haptic device. Experiments were conducted on microbeads instead of oocytes. The results confirmed that the time required for the experimental task was improved by 52.6%, and the injection error was improved by 75.3% compared to those observed in the conventional system.
Topics: Humans; Male; Sperm Injections, Intracytoplasmic; Haptic Interfaces; Semen; Infertility, Male; Oocytes; Spermatozoa
PubMed: 38276402
DOI: 10.3390/s24020711 -
Analytica Chimica Acta Apr 2024Opioids are effective painkillers used for medical purposes. Their prolonged ingestion can provoke some side effects (including overdose or constipation) that are...
BACKGROUND
Opioids are effective painkillers used for medical purposes. Their prolonged ingestion can provoke some side effects (including overdose or constipation) that are minimized by using opioid antagonists (e.g., naloxone). The rapid determination of opioids and their antagonists in biosamples is essential for an effective medical treatment. The direct combination of sample preparation and mass spectrometry (MS) fits well in this scenario. It can speed up the analysis achieving a good selectivity, which relies on the sample preparation and MS, and sensitivity levels.
RESULTS
This article presents a novel substrate-spray mass spectrometry interface based on a polydopamine-cotton (PDA-Cel) composite hosted inside the inner diameter of a 14-gauge blunt needle to determine oxycodone and naloxone in saliva samples. The needle is used as a microextraction device and a substrate for mass spectrometric analysis. The lack of sharpness of the 14-gauge (14G) blunt needles challenges the formation of the electrospray (ESI), and a commercial 10 μL pipette tip is proposed as a simple solution to this shortcoming. Under the optimum parameters, the proposed method was validated, obtaining limits of detection lower than 0.6 μg L, linear range up to 200 μg L, and linearity better than 0.9915. Relative standard deviation (RSD) and relative recoveries (RR) were studied at three different concentration levels (2, 40, and 200 μg L). RSD values were better than 20.7 %, and RR ranged from 90 to 114 %. Finally, a positive sample from a patient under medical treatment was analyzed.
SIGNIFICANCE AND NOVELTY
14G blunt needles have been demonstrated as effective extraction devices due to their low price (<0.15 € per extraction unit), their better safety (avoiding finger pricking), and their higher hosting capacity (up to 8 mg of sorbent). The conductivity of stainless steel permits their use as electrospray emitters, making their direct combination to MS easier. The large variety of fibrous sorbents makes this approach versatile enough to be adapted to other analytical problems.
Topics: Humans; Oxycodone; Naloxone; Saliva; Analgesics, Opioid; Mass Spectrometry
PubMed: 38438230
DOI: 10.1016/j.aca.2024.342376 -
PloS One 2024In remote communities, diagnosis of G6PD deficiency is challenging. We assessed the impact of modified test procedures and delayed testing for the point-of-care...
In remote communities, diagnosis of G6PD deficiency is challenging. We assessed the impact of modified test procedures and delayed testing for the point-of-care diagnostic STANDARD G6PD (SDBiosensor, RoK), and evaluated recommended cut-offs. We tested capillary blood from fingerpricks (Standard Method) and a microtainer (BD, USA; Method 1), venous blood from a vacutainer (BD, USA; Method 2), varied sample application methods (Methods 3), and used micropipettes rather than the test's single-use pipette (Method 4). Repeatability was assessed by comparing median differences between paired measurements. All methods were tested 20 times under laboratory conditions on three volunteers. The Standard Method and the method with best repeatability were tested in Indonesia and Nepal. In Indonesia 60 participants were tested in duplicate by both methods, in Nepal 120 participants were tested in duplicate by either method. The adjusted male median (AMM) of the Biosensor Standard Method readings was defined as 100% activity. In Indonesia, the difference between paired readings of the Standard and modified methods was compared to assess the impact of delayed testing. In the pilot study repeatability didn't differ significantly (p = 0.381); Method 3 showed lowest variability. One Nepalese participant had <30% activity, one Indonesian and 10 Nepalese participants had intermediate activity (≥30% to <70% activity). Repeatability didn't differ significantly in Indonesia (Standard: 0.2U/gHb [IQR: 0.1-0.4]; Method 3: 0.3U/gHb [IQR: 0.1-0.5]; p = 0.425) or Nepal (Standard: 0.4U/gHb [IQR: 0.2-0.6]; Method 3: 0.3U/gHb [IQR: 0.1-0.6]; p = 0.330). Median G6PD measurements by Method 3 were 0.4U/gHb (IQR: -0.2 to 0.7, p = 0.005) higher after a 5-hour delay compared to the Standard Method. The definition of 100% activity by the Standard Method matched the manufacturer-recommended cut-off for 70% activity. We couldn't improve repeatability. Delays of up to 5 hours didn't result in a clinically relevant difference in measured G6PD activity. The manufacturer's recommended cut-off for intermediate deficiency is conservative.
Topics: Humans; Male; Glucosephosphate Dehydrogenase; Pilot Projects; Sodium Oxybate; Glucosephosphate Dehydrogenase Deficiency; Biosensing Techniques
PubMed: 38241389
DOI: 10.1371/journal.pone.0296708 -
Novel Methods to Understand the Temporal Nature and Accuracy of Delivery for Insulin Infusion Pumps.Journal of Diabetes Science and... May 2024A wide suite of methods are available to evaluate delivery accuracy of insulin pumps. However, these methods do not capture any temporal information, which may be...
BACKGROUND
A wide suite of methods are available to evaluate delivery accuracy of insulin pumps. However, these methods do not capture any temporal information, which may be critical for design of artificial pancreas (AP) systems. We propose a novel video microscopy method to understand the delivery accuracy and temporal nature for a new durable pump under development (IFP), and a commercially available pump (Medtronic 722G, M722G).
METHODS
The cannula tip of an infusion set is inserted into a graduated pipette placed under a digital microscope. A video of the delivery is captured to track the fluid meniscus, to measure volumetric delivery rate and accuracy. This was done for a programmed value of 0.5 and 1 U. A similar procedure was adopted to track linear motion of the piston rod, which actuates the reservoir plunger, for a programmed value of 10 U.
RESULTS
It was observed that the commercially available pump delivers insulin in pulses of 0.05 U every two seconds. The mean absolute volumetric delivery error (MAE) for both pumps was found to be within the values reported previously. More importantly, it was found that a significant fraction of the programmed value is delivered, after completion of the planned bolus duration (IFP: 14.31% vs M722G: 9.38% for 1 U delivery).
CONCLUSIONS
The methods presented in this article help understand the delivery dynamics of liquid drug delivery devices. Our results indicate that a significant fraction of insulin delivery happens after the planned bolus duration, which might be important consideration for design of AP systems.
Topics: Insulin Infusion Systems; Humans; Insulin; Hypoglycemic Agents; Equipment Design; Video Recording; Time Factors
PubMed: 35929433
DOI: 10.1177/19322968221115749 -
Digital Discovery Oct 2023Closed-loop experiments can accelerate material discovery by automating both experimental manipulations and decisions that have traditionally been made by researchers....
Closed-loop experiments can accelerate material discovery by automating both experimental manipulations and decisions that have traditionally been made by researchers. Fast and non-invasive measurements are particularly attractive for closed-loop strategies. Viscosity is a physical property for fluids that is important in many applications. It is fundamental in application areas such as coatings; also, even if viscosity is not the key property of interest, it can impact our ability to do closed-loop experimentation. For example, unexpected increases in viscosity can cause liquid-handling robots to fail. Traditional viscosity measurements are manual, invasive, and slow. Here we use convolutional neural networks (CNNs) as an alternative to traditional viscometry by non-invasively extracting the spatiotemporal features of fluid motion under flow. To do this, we built a workflow using a dual-armed collaborative robot that collects video data of fluid motion autonomously. This dataset was then used to train a 3-dimensional convolutional neural network (3D-CNN) for viscosity estimation, either by classification or by regression. We also used these models to identify unknown laboratory solvents, again based on differences in fluid motion. The 3D-CNN model performance was compared with the performance of a panel of human participants for the same classification tasks. Our models strongly outperformed human classification in both cases. For example, even with training on fewer than 50 videos for each liquid, the 3D-CNN model gave an average accuracy of 88% for predicting the identity of five different laboratory solvents, compared to an average accuracy of 32% for human observation. For comparison, random category selection would give an average accuracy of 20%. Our method offers an alternative to traditional viscosity measurements for autonomous chemistry workflows that might be used both for process control (, choosing not to pipette liquids that are too viscous) or for materials discovery (, identifying new polymerization catalysts on the basis of viscosification).
PubMed: 38013903
DOI: 10.1039/d3dd00109a -
American Journal of Physiology. Cell... Jan 2024Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by Na1.7. We present evidence that this current is regulated by cAMP. ASM...
Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by Na1.7. We present evidence that this current is regulated by cAMP. ASM cells were isolated by enzymatic dispersal and studied using the whole cell patch clamp technique at room temperature. A fast sodium current, , was observed on holding cells under voltage clamp at -100 mV and stepping to -20 mV. This current was reduced in a concentration-dependent manner by denopamine (10 and 30 µM), a β-adrenergic agonist. Forskolin (1 µM), an activator of adenylate cyclase, reduced the current by 35%, but 6-MB-cAMP (300 µM), an activator of protein kinase A (PKA), had no effect. In contrast, 8-pCPT-2-O-Me-cAMP-AM (007-AM, 10 µM), an activator of exchange protein directly activated by cAMP (Epac), reduced the current by 48%. The inhibitory effect of 007-AM was still observed in the presence of dantrolene (10 µM), an inhibitor of ryanodine receptors, and when cytosolic [Ca] was buffered by inclusion of 1,2-bis(-aminophenoxy)ethane-,,,-tetraacetic acid, Sigma (BAPTA) (50 µM) in the pipette solution, suggesting that the inhibition of was not due to Ca-release from intracellular stores. When 007-AM was tested on the current-voltage relationship, it reduced the current at potentials from -30 to 0 mV, but had no effect on the steady-state activation curve. However, the steady-state inactivation , the voltage causing inactivation of 50% of the current, was shifted in the negative direction from -76.6 mV to -89.7 mV. These findings suggest that cAMP regulates in mouse ASM via Epac, but not PKA. β-adrenergic agonists are commonly used in inhalers to treat asthma and chronic obstructive pulmonary disease. These work by causing bronchodilation and reducing inflammation. The present study provides evidence that these drugs have an additional action, namely, to reduce sodium influx into airway smooth muscle cells via fast voltage-dependent channels. This may have the dual effect of promoting bronchodilation and reducing remodeling of the airways, which has a detrimental effect in these diseases.
Topics: Mice; Animals; Sodium; Cyclic AMP; Guanine Nucleotide Exchange Factors; Myocytes, Smooth Muscle; Adrenergic beta-Agonists
PubMed: 37955124
DOI: 10.1152/ajpcell.00417.2023