Biology subjects

Watts, B.

Publications and source records attributed to Watts, B..

6 recordsLinked to original sources

bioRxiv2026

Effect of Immunosuppressive Drugs on Glucose-Stimulated Insulin Secretion: Concentration-Response Studies in Dynamic Perifusion Assays

Immunosuppressive drugs, which are required to maintain graft function in transplant recipients, are associated with many unavoidable side effects including posttransplant diabetes mellitus (PTDM) that involves both peripheral insulin resistance and impairment of insulin secretion. To characterize in detail the concentration-dependency of the effect of well-known immunosuppressive drugs on glucose-stimulated insulin secretion (GSIS), we performed dynamic perifusion studies with human pancreatic islets. The effect on the time-profile of GSIS has been assessed over a wide concentration range for several clinically relevant immunomodulatory therapies, including small-molecule drugs (cyclosporine, sirolimus, tacrolimus, prednisolone acetate, and loteprednol etabonate) and biologics (abatacept and anti-CD40L), plus a prospective {beta}-cell proliferation-inducing agent (harmine). While biologics showed no significant detrimental effects after one-day treatment even at relatively high concentrations (5 {micro}M), all small-molecule drugs inhibited insulin secretion in a concentration-dependent manner, although glucocorticoids showed a distinct response pattern. Calcineurin and mTOR inhibitors preserved GSIS within their therapeutic ranges but progressively distorted its time-profile at higher concentrations and completely suppressed secretion at the highest levels. Cyclosporine exhibited the least, only about 35-fold, separation between its therapeutic target (Ctarg) and half-maximal GSIS inhibitory (IC50) concentrations. Glucocorticoids did not alter the shape of the time-profile but inhibited overall insulin secretion even at therapeutic levels. Their inhibitory effect only increased slowly with concentration and did not follow a classic sigmoid pattern that has unity Hill slope. These findings establish quantitative benchmarks for immunosuppressant-induced {beta}-cell toxicity and provide a framework for optimizing immunosuppressive regimens to reduce the risk of PTDM.

pharmacology and toxicology↗

bioRxiv2025

Evaluation of Ventilation at 10C as the Optimal Storage Condition for Donor Lungs in a Murine Model

RationaleCold static preservation at 4{degrees}C is the clinical standard for donor lung storage but is limited to 6-8 hours of cold ischemia. Static storage at 10{degrees}C has been shown to extend ischemia times and improve lung health. Given that lungs can maintain aerobic metabolism ex vivo, we hypothesized that adding ventilation at 10{degrees}C would further prolong preservation by stimulating aerobic metabolism. MethodsLungs were procured from C57Bl/6 mice and then stored for 24h with ventilation at 10{degrees}C (n=4), statically at 10{degrees}C (n=4), or statically at 4{degrees}C (n=4). Respiratory mechanics were evaluated using a FlexiVent system. Cellular viability was assessed via flow cytometry. Complement shedding was evaluated by enzyme-linked immunosorbent assay. Histologic evidence of lung injury was assessed by H&E staining. ResultsDonor lungs stored with ventilation at 10{degrees}C exhibited significantly reduced histologic injury scores compared to static storage at 4{degrees}C (p = 0.0062). Ventilation also decreased complement C3 shedding (p < 0.01), apoptosis (p < 0.05), cytochrome c release (p = 0.0014), and ROS production (p = 0.0008) compared to statically stored lungs at 4{degrees}C and 10{degrees}C. Functionally, ventilated lungs demonstrated improved respiratory mechanics with lower airway resistance (p = 0.021) and increased compliance (p = 0.023) compared to static storage at 10{degrees}C. ConclusionsVentilating lungs at 10{degrees}C compared to static cold storage appears to result in healthier and more functional lung tissue and may extend the preservation times of donor organs for lung transplantation.

physiology↗

bioRxiv2025

Assessing the Rate-Dependence of the First Phase of Glucose-Stimulated Insulin Secretion: Dynamic Perifusion Studies with Isolated Human Pancreatic Islets

Insulin released in response to a stepwise increase in glucose (square wave) is biphasic with a transient first-phase peak of 5-10 min and a more sustained second phase. While it is usually assumed that the first phase is rate- and the second is concentration-dependent, there are no detailed investigations into the rate-sensitivity of the first phase. We performed dynamic perifusion studies with isolated human pancreatic islets in a system that allowed fully customizable glucose ramps and established the corresponding insulin secretion time-profiles with high (once-a-minute) time-resolution. We considered the first-phase release to be the excess amount of insulin in addition to what would be expected from the glucose concentration-dependent second-phase release and examined its dependence on the glucose gradient (rate of increase). The average first-phase insulin release rate calculated this way increased with the gradient and could be fitted well with a Hill-type sigmoid function having a half-maximal value around 1.25 mM/min (nHill=1.8, r2=0.96). This agrees well with our previously introduced glucose-insulin control system built on a general framework of a sigmoid proportional-integral-derivative ({sigma}PID) controller, a generalized PID controller more suitable for biological systems than classic linear ones as biological responses are always limited between zero and a possible maximum. Experimental results obtained here were used to slightly readjust the parameters of this local glucose concentration-based computational model resulting in predictions in good agreement with measured first- and second-phase insulin secretions (r2>0.90). Thus, glucose-stimulated insulin secretion (GSIS) of perifused human islets can be described well as the sum of a rate-sensitive first phase, which is a sigmoid function of the glucose gradient with half-maximal value around 1.25 mM/min, and a concentration-sensitive second phase, which is a sigmoid function of the glucose concentration with half-maximal value around 8 mM.

physiology↗

bioRxiv2023

Visualizing alpha-synuclein and iron deposition in M83 mouse model of Parkinson's disease in vivo

BackgroundAbnormal alpha-synuclein and iron accumulation in the brain play an important role in Parkinsons disease (PD). Herein, we aim at visualizing alpha-synuclein inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. MethodsFluorescently labelled pyrimidoindole-derivative THK-565 was characterized by using recombinant fibrils and brains from 10-11 months old M83 mice, which subsequently underwent in vivo concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging. The in vivo results were verified against structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 Tesla and scanning transmission X-ray microscopy (STXM) of perfused brains. Brain slice immunofluorescence and Prussian blue staining were further performed to validate the detection of alpha-synuclein inclusions and iron deposition in the brain, respectively. ResultsTHK-565 showed increased fluorescence upon binding to recombinant alpha-synuclein fibrils and alpha-synuclein inclusions in post-mortem brain slices from patients with Parkinsons disease and M83 mice. i.v. administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 minutes post-injection by wide-field fluorescence compared to non-transgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. ConclusionWe demonstrated in vivo mapping of alpha-synuclein by means of non-invasive epifluorescence and vMSOT imaging assisted with a targeted THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.

neuroscience↗

bioRxiv2022

B cells discriminate HIV-1 Envelope protein affinities by sensing antigen binding association rates

HIV-1 Envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant, KD) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD, and whether B cells discriminate between proteins of similar affinities but that bind with different kinetic rates. Here we used a panel of Env proteins and Ramos B cell lines expressing IgM BCRs with specificity for CD4 binding-site broadly neutralizing (bnAb) or a precursor antibody to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD, but on sensing of association rate and a threshold antigen-BCR half-life.

immunology↗

bioRxiv2020

Cold sensitivity of the SARS-CoV-2 spike ectodomain

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible "up" or receptor-inaccessible "down" conformations. Here, we report that the commonly used stabilized S ectodomain construct "2P" is sensitive to cold temperature, and that this cold sensitivity is resolved in a "down" state stabilized spike. Our results will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.

biochemistry↗