Fluorescence to measure light intensity.

Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources.

Dynamics and interplay of photosynthetic regulatory processes depend on the amplitudes of oscillating light.

Plants have evolved multiple regulatory mechanisms to cope with natural light fluctuations. The interplay between these mechanisms leads presumably to the resilience of plants in diverse light patterns. We investigated the energy-dependent nonphotochemical quenching (qE) and cyclic electron transports (CET) in light that oscillated with a 60-s period with three different amplitudes. The photosystem I (PSI) and photosystem II (PSII) function-related quantum yields and redox changes of plastocyanin and ferredoxin were measured in Arabidopsis thaliana wild types and mutants with partial defects in qE or CET. The decrease in quantum yield of qE due to the lack of either PsbS- or violaxanthin de-epoxidase was compensated by an increase in the quantum yield of the constitutive nonphotochemical quenching. The mutant lacking NAD(P)H dehydrogenase (NDH)-like-dependent CET had a transient significant PSI acceptor side limitation during the light rising phase under high amplitude of light oscillations. The mutant lacking PGR5/PGRL1-CET restricted electron flows and failed to induce effective photosynthesis control, regardless of oscillation amplitudes. This suggests that PGR5/PGRL1-CET is important for the regulation of PSI function in various amplitudes of light oscillation, while NDH-like-CET acts' as a safety valve under fluctuating light with high amplitude. The results also bespeak interplays among multiple photosynthetic regulatory mechanisms.

Plants cope with fluctuating light by frequency-dependent nonphotochemical quenching and cyclic electron transport.

In natural environments, plants are exposed to rapidly changing light. Maintaining photosynthetic efficiency while avoiding photodamage requires equally rapid regulation of photoprotective mechanisms. We asked what the operation frequency range of regulation is in which plants can efficiently respond to varying light. Chlorophyll fluorescence, P700, plastocyanin, and ferredoxin responses of wild-types Arabidopsis thaliana were measured in oscillating light of various frequencies. We also investigated the npq1 mutant lacking violaxanthin de-epoxidase, the npq4 mutant lacking PsbS protein, and the mutants crr2-2, and pgrl1ab impaired in different pathways of the cyclic electron transport. The fastest was the PsbS-regulation responding to oscillation periods longer than 10 s. Processes involving violaxanthin de-epoxidase dampened changes in chlorophyll fluorescence in oscillation periods of 2 min or longer. Knocking out the PGR5/PGRL1 pathway strongly reduced variations of all monitored parameters, probably due to congestion in the electron transport. Incapacitating the NDH-like pathway only slightly changed the photosynthetic dynamics. Our observations are consistent with the hypothesis that nonphotochemical quenching in slow light oscillations involves violaxanthin de-epoxidase to produce, presumably, a largely stationary level of zeaxanthin. We interpret the observed dynamics of photosystem I components as being formed in slow light oscillations partially by thylakoid remodeling that modulates the redox rates.

Effect of organic photovoltaic and red-foil transmittance on yield, growth and photosynthesis of two spinach genotypes under field and greenhouse conditions.

The galloping rise in global population in recent years and the accompanying increase in food and energy demands has created land use crisis between food and energy production, and eventual loss of agricultural lands to the more lucrative photovoltaics (PV) energy production. This experiment was carried out to investigate the effect of organic photovoltaics (OPV) and red-foil (RF) transmittance on growth, yield, photosynthesis and SPAD value of spinach under greenhouse and field conditions. Three OPV levels (P0: control; P1: transmittance peak of 0.11 in blue light (BL) and 0.64 in red light (RL); P2: transmittance peak of 0.09 in BL and 0.11 in RL) and two spinach genotypes (bufflehead, eland) were combined in a 3 × 2 factorial arrangement in a completely randomized design with 4 replications in the greenhouse, while two RF levels (RF0: control; RF1: transmittance peak of 0.01 in BL and 0.89 in RL) and two spinach genotypes were combined in a 2 × 2 factorial in randomized complete block design with four replications in the field. Data were collected on growth, yield, photosynthesis and chlorophyll content. Analysis of variance (ANOVA) showed significant reduction in shoot weight and total biomass of spinach grown under very low light intensities as a function of the transmittance properties of the OPV cell used (P2). P1 competed comparably (p > 0.05) with control in most growth and yield traits measured. In addition, shoot to root distribution was higher in P1 than control. RF reduced shoot and total biomass production of spinach in the field due to its inability to transmit other spectra of light. OPV-RF transmittance did not affect plant height (PH), leaf number (LN), and SPAD value but leaf area (LA) was highest in P2. Photochemical energy conversion was higher in P1, P2 and RF1 in contrast to control due to lower levels of non-photochemical energy losses through the Y(NO) and Y(NPQ) pathways. Photo-irradiance curves showed that plants grown under reduced light (P2) did not efficiently manage excess light when exposed to high light intensities. Bufflehead genotype showed superior growth and yield traits than eland across OPV and RF levels. It is therefore recommended that OPV cells with transmittance properties greater than or equal to 11% in BL and 64% in RL be used in APV systems for improved photochemical and land use efficiency.

Insights on the regulation of photosynthesis in pea leaves exposed to oscillating light.

Plants growing in nature often experience fluctuating irradiance. However, in the laboratory, the dynamics of photosynthesis are usually explored by instantaneously exposing dark-adapted plants to constant light and examining the dark-to-light transition, which is a poor approximation of natural phenomena. With the aim creating a better approximation, we exposed leaves of pea (Pisum sativum) to oscillating light and measured changes in the functioning of PSI and PSII, and of the proton motive force at the thylakoid membrane. We found that the dynamics depended on the oscillation period, revealing information about the underlying regulatory networks. As demonstrated for a selected oscillation period of 60 s, the regulation tries to keep the reaction centers of PSI and PSII open. We present an evaluation of the data obtained, and discuss the involvement of particular processes in the regulation of photosynthesis. The forced oscillations provided an information-rich fingerprint of complex regulatory networks. We expect future progress in understanding these networks from experiments involving chemical interventions and plant mutants, and by using mathematical modeling and systems identification and control tools.

The Kok effect revisited.

The Kok effect refers to the abrupt decrease around the light compensation point in the slope of net photosynthetic rate vs irradiance. Arguably, this switch arises from light inhibition of respiration, allowing the Kok method to estimate day respiration (Rd ). Recent analysis suggests that increasing proportions of photorespiration (quantified as Γ*/Cc , the ratio of CO2 compensation point Γ* to chloroplast CO2 concentration, Cc ) with irradiance explain much of the Kok effect. Also, the Kok method has been modified to account for the decrease in PSII photochemical efficiency (Φ2 ) with irradiance. Using a model that illustrates how varying Rd , Γ*/Cc , Φ2 and proportions of alternative electron transport could engender the Kok effect, we quantified the contribution of these parameters to the Kok effect measured in sunflower across various O2 and CO2 concentrations and various temperatures. Overall, the decreasing Φ2 with irradiance explained c. 12%, and the varying Γ*/Cc explained c. 25%, of the Kok effect. Maximum real light inhibition of Rd was much lower than the inhibition derived from the Kok method, but still increased with photorespiration. Photorespiration had a dual contribution to the Kok effect, one via the varying Γ*/Cc and the other via its participation in light inhibition of Rd .

Misshapen/NIK-related kinase (MINK1) is involved in platelet function, hemostasis, and thrombus formation.

The sterile-20 kinase misshapen/Nck-interacting kinase (NIK)-related kinase 1 (MINK1) is involved in many important cellular processes such as growth, cytoskeletal rearrangement, and motility. Here, with MINK1-deficient (MINK1(-/-)) mice, we showed that MINK1 plays an important role in hemostasis and thrombosis via the regulation of platelet functions. In the tail-bleeding assay, MINK1(-/-) mice exhibited a longer bleeding time than wild-type (WT) mice (575.2 ± 59.7 seconds vs 419.6 ± 66.9 seconds). In a model of ferric chloride-induced mesenteric arteriolar thrombosis, vessel occlusion times were twice as long in MINK1(-/-) mice as in WT mice. In an in vitro microfluidic whole-blood perfusion assay, thrombus formation on a collagen matrix under arterial shear conditions was significantly reduced in MINK1(-/-) platelets. Moreover, MINK1(-/-) platelets demonstrated impaired aggregation and secretion in response to low doses of thrombin and collagen. Furthermore, platelet spreading on fibrinogen was largely hampered in MINK1(-/-) platelets. The functional differences of MINK1(-/-) platelets could be attributed to impaired adenosine 5'-diphosphate secretion. Signaling events associated with MINK1 appeared to involve extracellular signal-regulated kinase, p38, and Akt. Hence, MINK1 may be an important signaling molecule that mediates mitogen-activated protein kinase signaling and participates in platelet activation and thrombus formation.

Platelets promote cartilage repair and chondrocyte proliferation via ADP in a rodent model of osteoarthritis.

Osteoarthritis (OA) is the most common age-related degenerative joint disease and platelet-rich plasma (PRP) has been shown to be beneficial in OA. Therefore, in this study, we aimed to investigate the effects of platelets on chondrocytes and the underlying mechanisms. Anabolic and catabolic activity and the proliferation rate of chondrocytes were evaluated after co-culture with platelets. Chondrocyte gene expression was measured by real-time PCR. Chondrocyte protein expression and phosphorylation were measured by western blot. Chondrocytes treated with or without platelets were transplanted into a rat model of OA induced by intra-articular injection of monosodium iodoacetate and the repair of articular cartilage was evaluated macroscopically and histologically. Platelets significantly promoted the proliferation of chondrocytes, while mildly influencing anabolic and catabolic activity. Chondrocytes co-cultured with platelets showed significantly increased production of bone morphogenetic protein 7 (BMP7). The autocrine/paracrine effect of BMP7 was responsible for the increased proliferation of chondrocytes, via the ERK/CDK1/cyclin B1 signaling pathway. Transplantation of platelet-treated chondrocytes showed better cartilage repair in the OA model. Platelet-derived ADP was identified as the major mediator to promote the production of BMP7 and the proliferation of chondrocytes, through the ADP receptor P2Y1. Finally, direct injection of α,ß-methyleneadenosine-5'-diphosphate into OA joints also enhanced cartilage repair. This study has identified that platelet-derived ADP, but not ATP, is the key mediator for platelet-promoted chondrocyte proliferation and cartilage repair in osteoarthritis. This finding may provide a key explanation for the therapeutic effect of platelets in OA and help shaping a strategy to improve OA therapy.

Codominant IgG and IgA expression with minimal vaccine mRNA in milk of BNT162b2 vaccinees.

Lactating women can produce protective antibodies in their milk after vaccination, which has informed antenatal vaccination programs for diseases such as influenza and pertussis. However, whether SARS-CoV-2-specific antibodies are produced in human milk as a result of COVID-19 vaccination is still unclear. In this study, we show that lactating mothers who received the BNT162b2 vaccine secreted SARS-CoV-2-specific IgA and IgG antibodies into milk, with the most significant increase at 3-7 days post-dose 2. Virus-specific IgG titers were stable out to 4-6 weeks after dose 2. In contrast, SARS-CoV-2-specific IgA levels showed substantial decay. Vaccine mRNA was detected in few milk samples (maximum of 2 ng/ml), indicative of minimal transfer. Additionally, infants who consumed post-vaccination human milk had no reported adverse effects up to 28 days post-ingestion. Our results define the safety and efficacy profiles of the vaccine in this demographic and provide initial evidence for protective immunity conferred by milk-borne SARS-CoV-2-specific antibodies. Taken together, our study supports recommendations for uninterrupted breastfeeding subsequent to mRNA vaccination against COVID-19.

Google Glass-Directed Monitoring and Control of Microfluidic Biosensors and Actuators.

Google Glass is a recently designed wearable device capable of displaying information in a smartphone-like hands-free format by wireless communication. The Glass also provides convenient control over remote devices, primarily enabled by voice recognition commands. These unique features of the Google Glass make it useful for medical and biomedical applications where hands-free experiences are strongly preferred. Here, we report for the first time, an integral set of hardware, firmware, software, and Glassware that enabled wireless transmission of sensor data onto the Google Glass for on-demand data visualization and real-time analysis. Additionally, the platform allowed the user to control outputs entered through the Glass, therefore achieving bi-directional Glass-device interfacing. Using this versatile platform, we demonstrated its capability in monitoring physical and physiological parameters such as temperature, pH, and morphology of liver- and heart-on-chips. Furthermore, we showed the capability to remotely introduce pharmaceutical compounds into a microfluidic human primary liver bioreactor at desired time points while monitoring their effects through the Glass. We believe that such an innovative platform, along with its concept, has set up a premise in wearable monitoring and controlling technology for a wide variety of applications in biomedicine.