The use of hyperbaric oxygen therapy in the treatment of hand crush injuries.

Hyperbaric oxygen therapy (HBOT) has been used as an adjuvant treatment for crush injury because it can improve tissue hypoxia and stimulate wound healing. However, the actual role of HBOT in crush hand injury is still unknown. This study is to assess the efficacy of HBOT for crush hand patients, as well as the impact of HBOT initiation timing. Between 2018 and 2021, 72 patients with crush hand injury were retrospectively reviewed. The patients were divided into the HBOT and control group, and each group had 36 patients. The average session of HBOT was 18.2 (5-32 sessions) per patient, and no patient had a complication related to the treatment. The two groups had similar demographics, but HBOT group had larger injured area (73.6 ± 51.0 vs. 48.2 ± 45.5 cm2 , p = 0.03). To better control the confounding factors, we performed the subgroup analysis with cut-off injured area of 50 cm2 . In the patients with smaller injured area (≦50 cm2 ), the HBOT group had shorter wound healing time (29.9 ± 12.9 vs. 41.0 ± 18.9 days, p = 0.03). The early HBOT group (first session ≤72 h post-operatively) had shorter hospital stay (8.1 ± 6.4 vs. 15.5 ± 11.4 days, p = 0.04), faster wound healing (28.7 ± 17.8 vs. 41.1 ± 18.1 days, p = 0.08) and less operations (1.54 ± 0.78 vs. 2.41 ± 1.62, p = 0.06) although the latter two didn't achieve statistical significance. HBOT is safe and effective in improving wound healing of hand crush injury. Early intervention of HBOT may be more beneficial. Future research is required to provide more evidence.

Measurements of absolute line strength of the ν1 fundamental transitions of OH radical and rate coefficient of the reaction OH + H2O2 with mid-infrared two-color time-resolved dual-comb spectroscopy.

Absolute line strengths of several transitions in the ν1 fundamental band of the hydroxyl radical (OH) have been measured by simultaneous determination of hydrogen peroxide (H2O2) and OH upon laser photolysis of H2O2. Based on the well-known quantum yield for the generation of OH radicals in the 248-nm photolysis of H2O2, the line strength of the OH radicals can be accurately derived by adopting the line strength of the well-characterized transitions of H2O2 and analyzing the difference absorbance time traces of H2O2 and OH obtained upon laser photolysis. Employing a synchronized two-color dual-comb spectrometer, we measured high-resolution time-resolved absorption spectra of H2O2 near 7.9 µm and the OH radical near 2.9 µm, simultaneously, under varied conditions. In addition to the studies of the line strengths of the selected H2O2 and OH transitions, the kinetics of the reaction between OH and H2O2 were investigated. A pressure-independent rate coefficient kOH+H2O2 was determined to be [1.97 (+0.10/-0.15)] × 10-12 cm3 molecule-1 s-1 at 296 K and compared with other experimental results. By carefully analyzing both high-resolution spectra and temporal absorbance profiles of H2O2 and OH, the uncertainty of the obtained OH line strengths can be achieved down to <10% in this work. Moreover, the proposed two-color time-resolved dual-comb spectroscopy provides a new approach for directly determining the line strengths of transient free radicals and holds promise for investigations on their self-reaction kinetics as well as radical-radical reactions.

Synaptophysin Regulates Fusion Pores and Exocytosis Mode in Chromaffin Cells.

Synaptophysin (syp) is a major integral membrane protein of secretory vesicles. Previous work has demonstrated functions for syp in synaptic vesicle cycling, endocytosis, and synaptic plasticity, but the role of syp in the process of membrane fusion during Ca2+-triggered exocytosis remains poorly understood. Furthermore, although syp resides on both large dense-core and small synaptic vesicles, its role in dense-core vesicle function has received less attention compared with synaptic vesicle function. To explore the role of syp in membrane fusion and dense-core vesicle function, we used amperometry to measure catecholamine release from single vesicles in male and female mouse chromaffin cells with altered levels of syp and the related tetraspanner protein synaptogyrin (syg). Knocking out syp slightly reduced the frequency of vesicle fusion events below wild-type (WT) levels, but knocking out both syp and syg reduced the frequency 2-fold. Knocking out both proteins stabilized initial fusion pores, promoted fusion pore closure (kiss-and-run), and reduced late-stage fusion pore expansion. Introduction of a syp construct lacking its C-terminal dynamin-binding domain in syp knock-outs (KOs) increased the duration and fraction of kiss-and-run events, increased total catecholamine release per event, and reduced late-stage fusion pore expansion. These results demonstrated that syp and syg regulate dense-core vesicle function at multiple stages to initiate fusion, control the choice of mode between full-fusion and kiss-and-run, and influence the dynamics of both initial and late-stage fusion pores. The transmembrane domain (TMD) influences small initial fusion pores, and the C-terminal domain influences large late-stage fusion pores, possibly through an interaction with dynamin.SIGNIFICANCE STATEMENT The secretory vesicle protein synaptophysin (syp) is known to function in synaptic vesicle cycling, but its roles in dense-core vesicle functions, and in controlling membrane fusion during Ca2+-triggered exocytosis remain unclear. The present study used amperometry recording of catecholamine release from endocrine cells to assess the impact of syp and related proteins on membrane fusion. A detailed analysis of amperometric spikes arising from the exocytosis of single vesicles showed that these proteins influence fusion pores at multiple stages and control the choice between kiss-and-run and full-fusion. Experiments with a syp construct lacking its C terminus indicated that the transmembrane domain (TMD) influences the initial fusion pore, while the C-terminal domain influences later stages after fusion pore expansion.

Electrically Controllable Kondo Correlation in Spin-Orbit-Coupled Quantum Point Contacts.

Integrating the Kondo correlation and spin-orbit interactions, each of which have individually offered unprecedented means to manipulate electron spins, in a controllable way can open up new possibilities for spintronics. We demonstrate electrical control of the Kondo correlation by coupling the bound spin to leads with tunable Rashba spin-orbit interactions, realized in semiconductor quantum point contacts. We observe a transition from single to double peak zero-bias anomalies in nonequilibrium transport-the manifestation of the Kondo effect-indicating a controlled Kondo spin reversal using only spin-orbit interactions. Universal scaling of the Kondo conductance is demonstrated, implying that the spin-orbit interactions could enhance the Kondo temperature. A theoretical model based on quantum master equations is also developed to calculate the nonequilibrium quantum transport.

Physical geography, isolation by distance and environmental variables shape genomic variation of wild barley (Hordeum vulgare L. ssp. spontaneum) in the Southern Levant.

Determining the extent of genetic variation that reflects local adaptation in crop-wild relatives is of interest for the purpose of identifying useful genetic diversity for plant breeding. We investigated the association of genomic variation with geographical and environmental factors in wild barley (Hordeum vulgare L. ssp. spontaneum) populations of the Southern Levant using genotyping by sequencing (GBS) of 244 accessions in the Barley 1K+ collection. The inference of population structure resulted in four genetic clusters that corresponded to eco-geographical habitats and a significant association between lower gene flow rates and geographical barriers, e.g. the Judaean Mountains and the Sea of Galilee. Redundancy analysis (RDA) revealed that spatial autocorrelation explained 45% and environmental variables explained 15% of total genomic variation. Only 4.5% of genomic variation was solely attributed to environmental variation if the component confounded with spatial autocorrelation was excluded. A synthetic environmental variable combining latitude, solar radiation, and accumulated precipitation explained the highest proportion of genomic variation (3.9%). When conditioned on population structure, soil water capacity was the most important environmental variable explaining 1.18% of genomic variation. Genome scans with outlier analysis and genome-environment association studies were conducted to identify adaptation signatures. RDA and outlier methods jointly detected selection signatures in the pericentromeric regions, which have reduced recombination, of the chromosomes 3H, 4H, and 5H. However, selection signatures mostly disappeared after correction for population structure. In conclusion, adaptation to the highly diverse environments of the Southern Levant over short geographical ranges had a limited effect on the genomic diversity of wild barley. This highlighted the importance of nonselective forces in genetic differentiation.

A pressure ulcers assessment system for diagnosis and decision making using convolutional neural networks.

BACKGROUND/PURPOSE: Pressure ulcers are a common problem in hospital care and long-term care. Pressure ulcers are caused by prolonged compression of soft tissues, which can cause local tissue damage and even lead to serious infections. This study uses a deep learning algorithm to construct a system that diagnoses pressure ulcers and assists in making treatment decisions, thus providing additional reference for first-line caregivers. METHODS: We performed a retrospective research of medical records to find photos of patients with pressure ulcers at National Taiwan University Hospital from 2016 to 2020. We used photos from 2016 to 2019 for training and after removing the photos which were vague, underexposed, or overexposed, 327 photos were obtained. The photos were then labeled as "erythema" or "non-erythema" for the first classification task and "extensive necrosis", "moderate necrosis" or "limited necrosis" for the second, by consensus of three recruited physicians. An Inception-ResNet-v2 model, a kind of Convolutional Neural Network (CNN), was applied for training these two classification tasks to construct an assessment system. Finally, we tested the model with the photos of pressure ulcers taken from 2019 to 2020 to verify its accuracy. RESULTS: For the task of classification of erythema and non-erythema wounds, our CNN model achieved an accuracy of about 98.5%. For the task of classification of necrotic tissue, our model achieved accuracy of about 97%. CONCLUSION: Our CNN model, which was based on Inception-ResNet-v2, achieved high accuracy when classifying different types of pressure ulcers, making it applicable in clinical circumstances.

Comparison of the Forward and Reverse Photocycle Dynamics of Two Highly Similar Canonical Red/Green Cyanobacteriochromes Reveals Unexpected Differences.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that exhibit photochromism between two states: a thermally stable dark-adapted state and a metastable light-adapted state with bound linear tetrapyrrole (bilin) chromophores possessing 15Z and 15E configurations, respectively. The photodynamics of canonical red/green CBCRs have been extensively studied; however, the time scales of their excited-state lifetimes and subsequent ground-state evolution rates widely differ and, at present, remain difficult to predict. Here, we compare the photodynamics of two closely related red/green CBCRs that have substantial sequence identity (∼68%) and similar chromophore environments: AnPixJg2 from Anabaena sp. PCC 7120 and NpR6012g4 from Nostoc punctiforme. Using broadband transient absorption spectroscopy on the primary (125 fs to 7 ns) and secondary (7 ns to 10 ms) time scales together with global analysis modeling, our studies revealed that AnPixJg2 and NpR6012g4 have comparable quantum yields for initiating the forward (15ZPr → 15EPg) and reverse (15EPg → 15ZPr) reactions, which proceed through monotonic and nonmonotonic mechanisms, respectively. In addition to small discrepancies in the kinetics, the secondary reverse dynamics resolved unique features for each domain: intermediate shunts in NpR6012g4 and a Meta-Gf intermediate red-shifted from the 15ZPr photoproduct in AnPixJg2. Overall, this study supports the conclusion that sequence similarity is a useful criterion for predicting pathways of the light-induced evolution and quantum yield of generating primary intermediate Φp within subfamilies of CBCRs, but more studies are still needed to develop a comprehensive molecular level understanding of these processes.

An intrinsic, label-free signal for identifying stem cell-derived cardiomyocyte subtype.

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have many promising applications, including the regeneration of injured heart muscles, cardiovascular disease modeling, and drug cardiotoxicity screening. Current differentiation protocols yield a heterogeneous cell population that includes pluripotent stem cells and different cardiac subtypes (pacemaking and contractile cells). The ability to purify these cells and obtain well-defined, controlled cell compositions is important for many downstream applications; however, there is currently no established and reliable method to identify hiPSC-derived cardiomyocytes and their subtypes. Here, we demonstrate that second harmonic generation (SHG) signals generated directly from the myosin rod bundles can be a label-free, intrinsic optical marker for identifying hiPSC-derived cardiomyocytes. A direct correlation between SHG signal intensity and cardiac subtype is observed, with pacemaker-like cells typically exhibiting ~70% less signal strength than atrial- and ventricular-like cardiomyocytes. These findings suggest that pacemaker-like cells can be separated from the heterogeneous population by choosing an SHG intensity threshold criteria. This work lays the foundation for developing an SHG-based high-throughput flow sorter for purifying hiPSC-derived cardiomyocytes and their subtypes.

Human induced pluripotent stem cell line with genetically encoded fluorescent voltage indicator generated via CRISPR for action potential assessment post-cardiogenesis.

Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.

Comparative analysis of patients with upper urinary tract urothelial carcinoma in black-foot disease endemic and non-endemic area.

BACKGROUND: A high incidence of upper urinary tract urothelial carcinoma has been reported in the southwestern area of Taiwan, where arsenic water contamination was considered the main cause. However, there is no definite proof to show a correlation between arsenic water contamination and upper urinary tract urothelial carcinoma. To investigate the clinical and epidemiological features of patients with upper urinary tract urothelial carcinoma between arsenic water endemic and non-endemic areas, we analyzed patients in terms of characteristics, stratified overall survival, disease-free survival, and cancer-specific survival. METHODS: The records of a total of 1194 patients diagnosed with upper urinary tract urothelial carcinoma were retrospectively reviewed. Clinical data and current medical status were collected from the medical records. Statistical analyses were performed to determine the clinical variables and stratified survival curves between endemic and non-endemic groups. RESULTS: Female predominance was revealed in both endemic and non-endemic groups (male:female ratio = 1:1.2-1.4). No statistical differences were found in histological types, staging, and tumor size between the two groups. Nonetheless, patients with characteristics of aging and having end-stage renal disease were outnumbered in the non-endemic group, while a higher prevalence of previous bladder tumors and more ureteral tumors were found in the endemic group. Adjusted stratified cumulative survival curves suggested a poorer prognosis in endemic patients, especially in disease-free survival of early stage disease. CONCLUSIONS: A higher mortality rate with more previous bladder cancer history and ureteral tumors was seen in patients with upper urinary tract urothelial carcinoma residing in the arsenic water contamination area. This may be attributed to the long-term carcinogenic effect of arsenic underground water.

Correlating structural and photochemical heterogeneity in cyanobacteriochrome NpR6012g4.

Phytochrome photoreceptors control plant growth, development, and the shade avoidance response that limits crop yield in high-density agricultural plantings. Cyanobacteriochromes (CBCRs) are distantly related photosensory proteins that control cyanobacterial metabolism and behavior in response to light. Photoreceptors in both families reversibly photoconvert between two photostates via photoisomerization of linear tetrapyrrole (bilin) chromophores. Spectroscopic and biochemical studies have demonstrated heterogeneity in both photostates, but the structural basis for such heterogeneity remains unclear. We report solution NMR structures for both photostates of the red/green CBCR NpR6012g4 from Nostoc punctiforme In addition to identifying structural changes accompanying photoconversion, these structures reveal structural heterogeneity for residues Trp655 and Asp657 in the red-absorbing NpR6012g4 dark state, yielding two distinct environments for the phycocyanobilin chromophore. We use site-directed mutagenesis and fluorescence and absorbance spectroscopy to assign an orange-absorbing population in the NpR6012g4 dark state to the minority configuration for Asp657. This population does not undergo full, productive photoconversion, as shown by time-resolved spectroscopy and absorption spectroscopy at cryogenic temperature. Our studies thus elucidate the spectral and photochemical consequences of structural heterogeneity in a member of the phytochrome superfamily, insights that should inform efforts to improve photochemical or fluorescence quantum yields in the phytochrome superfamily.

Using Dorsal Metacarpal Artery Perforator Flap in the Soft Tissue Reconstruction of Traumatic Finger Defects: A Single-Center Study.

INTRODUCTION: The reconstruction of soft tissue defects of fingers is a challenge due to the limitations of local tissue availability. The dorsal metacarpal artery perforator (DMAP) flap is a vascular island flap raised on the dorsum of the hand, and it is a good option for finger reconstruction by replacing similar-for-similar in a single operation. In this study, we would like to share our experience of using the DMAP flap in cases of various traumatic finger defects. MATERIALS AND METHODS: From November 2016 to May 2019, patients who had traumatic finger injuries and had undergone DMAP flap for soft tissue reconstruction were examined. The patients' demographic data, injury and flap characteristics, wound healing status, and complications were collected and studied. The functional and aesthetic outcomes were evaluated using the Michigan Hand Outcomes Questionnaire. RESULTS: There was a total of 10 patients included in this study, of which 9 were male and 1 was female. The average age was 43 years (17-66 years). Seven patients were administered general anesthesia, and 3 others wide-awake local anesthesia. The average flap size was 4.9 × 2.0 cm, and all the donor sites were primarily closed. Nearly half of the patients had temporary venous congestion, but most of the flaps survived well ultimately. Only 1 patient had a partial flap necrosis, which required an additional skin graft. CONCLUSIONS: The DMAP flap offers a thin and pliable skin to reconstruct finger defects within 1-stage surgery. It is easy to harvest with reliable and constant circulation. With adequate design, the DMAP flap can be used to resurface both volar and dorsal finger defects and also can reach the tip of the little finger. The DMAP flap is the ideal flap for reconstruction of traumatic finger defect with either local or general anesthesia.

Fusion Pore Expansion and Contraction during Catecholamine Release from Endocrine Cells.

Amperometry recording reveals the exocytosis of catecholamine from individual vesicles as a sequential process, typically beginning slowly with a prespike foot, accelerating sharply to initiate a spike, reaching a peak, and then decaying. This complex sequence reflects the interplay between diffusion, flux through a fusion pore, and possibly dissociation from a vesicle's dense core. In an effort to evaluate the impacts of these factors, a model was developed that combines diffusion with flux through a static pore. This model accurately recapitulated the rapid phase of a spike but generated relations between spike shape parameters that differed from the relations observed experimentally. To explore the possible role of fusion pore dynamics, a transformation of amperometry current was introduced that yields fusion pore permeability divided by vesicle volume (g/V). Applying this transform to individual fusion events yielded a highly characteristic time course. g/V initially tracks the current, increasing ∼15-fold from the prespike foot to the spike peak. After the peak, g/V unexpectedly declines and settles into a plateau that indicates the presence of a stable postspike pore. g/V of the postspike pore varies greatly between events and has an average that is ∼3.5-fold below the peak value and ∼4.5-fold above the prespike value. The postspike pore persists and is stable for tens of milliseconds, as long as catecholamine flux can be detected. Applying the g/V transform to rare events with two peaks revealed a stepwise increase in g/V during the second peak. The g/V transform offers an interpretation of amperometric current in terms of fusion pore dynamics and provides a, to our knowledge, new frameworkfor analyzing the actions of proteins that alter spike shape. The stable postspike pore follows from predictions of lipid bilayer elasticity and offers an explanation for previous reports of prolonged hormone retention within fusing vesicles.

Conservation and Diversity in the Primary Reverse Photodynamics of the Canonical Red/Green Cyanobacteriochrome Family.

In this report, we compare the femtosecond to nanosecond primary reverse photodynamics (15EPg → 15ZPr) of eight tetrapyrrole binding photoswitching cyanobacteriochromes in the canonical red/green family from the cyanobacterium Nostoc punctiforme. Three characteristic classes were identified on the basis of the diversity of excited-state and ground-state properties, including the lifetime, photocycle initiation quantum yield, photointermediate stability, spectra, and temporal properties. We observed a correlation between the excited-state lifetime and peak wavelength of the electronic absorption spectrum with higher-energy-absorbing representatives exhibiting both faster excited-state decay times and higher photoisomerization quantum yields. The latter was attributed to both an increased number of structural restraints and differences in H-bonding networks that facilitate photoisomerization. All three classes exhibited primary Lumi-Go intermediates, with class II and III representatives evolving to a secondary Meta-G photointermediate. Class II Meta-GR intermediates were orange absorbing, whereas class III Meta-G had structurally relaxed, red-absorbing chromophores that resemble their dark-adapted 15ZPr states. Differences in the reverse and forward reaction mechanisms are discussed within the context of structural constraints.

Surface-enhanced Raman scattering sensing platform for detecting amyloid-ß peptide interaction with an aggregation inhibitor.

Soluble, small amyloid-ß oligomers (AßO) are recognized as significant contributors to the pathology of Alzheimer's disease (AD). Although drugs for treating AD symptoms have been approved, no therapy targeting amyloid-ß (Aß) capable of modifying the course of the disease is available. In an effort to develop a label-free method for screening new anti-AD therapeutic agents, we show the use of a surface-enhanced Raman scattering (SERS) active substrate for detecting the interactions between Aß peptides and spin-labeled fluorine (SLF), a peptide aggregation inhibitor. Changes in the peak positions and intensity ratios of two spectral peaks near 1600cm-1 and 2900cm-1 can be used to monitor the molecular interactions between SLF and Aß. This study demonstrates the potential of SERS spectroscopy for rapidly screening and identifying new anti-Aß therapeutic agents.

An atypical hyperosmolar hyperglycemic state and diabetic ketoacidosis induced by sodium-glucose cotransporter-2 inhibitors: A case report.

Sodium-glucose cotransporter-2 (SGLT-2) inhibitors have become the best choice of second-line oral antidiabetic drugs for patients with heart or chronic kidney disease. However, it is not clear how safe this treatment is for elderly patients, especially Asians, who are known to be more insulin deficient than Caucasian individuals with a similar insulin resistance. Here, we report a case concerning an elderly patient with type 2 diabetes mellitus without insulin dependence, whose antidiabetic medication had recently been changed to include an SGLT-2 inhibitor. The patient presented with an atypical hyperosmolar hyperglycemic state and diabetic ketoacidosis but recovered after insulin pump treatment and fluid supplementation. The patient was discharged with a prescription of a mixed-type insulin injection instead of oral antidiabetic medications for diabetes control. Our case demonstrates that if SGLT-2 inhibitors are administered to elderly Asian patients, their benefits and adverse effects should be carefully monitored.

The Transmembrane Domain of Synaptobrevin Influences Neurotransmitter Flux through Synaptic Fusion Pores.

The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins synaptobrevin (Syb), syntaxin, and SNAP-25 function in Ca2+-triggered exocytosis in both endocrine cells and neurons. The transmembrane domains (TMDs) of Syb and syntaxin span the vesicle and plasma membrane, respectively, and influence flux through fusion pores in endocrine cells as well as fusion pores formed during SNARE-mediated fusion of reconstituted membranes. These results support a model for exocytosis in which SNARE TMDs form the initial fusion pore. The present study sought to test this model in synaptic terminals. Patch-clamp recordings of miniature EPSCs (mEPSCs) were used to probe fusion pore properties in cultured hippocampal neurons from mice of both sexes. Mutants harboring tryptophan at four different sites in the Syb TMD reduced the rate-of-rise of mEPSCs. A computer model that simulates glutamate diffusion and receptor activation kinetics could account for this reduction in mEPSC rise rate by slowing the flux of glutamate through synaptic fusion pores. TMD mutations introducing positive charge also reduced the mEPSC rise rate, but negatively charged residues and glycine, which should have done the opposite, had no effect. The sensitivity of mEPSCs to pharmacological blockade of receptor desensitization was enhanced by a mutation that slowed the mEPSC rate-of-rise, suggesting that the mutation prolonged the residence of glutamate in the synaptic cleft. The same four Syb TMD residues found here to influence synaptic release were found previously to influence endocrine release, leading us to propose that a similar TMD-lined fusion pore functions widely in Ca2+-triggered exocytosis in mammalian cells.SIGNIFICANCE STATEMENT SNARE proteins function broadly in biological membrane fusion. Evidence from non-neuronal systems suggests that SNARE proteins initiate fusion by forming a fusion pore lined by transmembrane domains, but this model has not yet been tested in synapses. The present study addressed this question by testing mutations in the synaptic vesicle SNARE synaptobrevin for an influence on the rise rate of miniature synaptic currents. These results indicate that synaptobrevin's transmembrane domain interacts with glutamate as it passes through the fusion pore. The sites in synaptobrevin that influence this flux are identical to those shown previously to influence flux through endocrine fusion pores. Thus, SNARE transmembrane domains may function in the fusion pores of Ca2+-triggered exocytosis of both neurotransmitters and hormones.

Forward Photodynamics of the Noncanonical Red/Green NpR3784 Cyanobacteriochrome from Nostoc punctiforme.

Cyanobacteriochromes (CBCRs) make up a diverse family of cyanobacterial photoreceptors distantly related to the phytochrome photoreceptors of land plants. At least two lineages of CBCRs have reacquired red-absorbing dark states similar to the phytochrome Pr resting state but are coupled to green-absorbing light-adapted states rather than the canonical far-red-absorbing light-adapted state. One such lineage includes the canonical red/green (R/G) CBCRs that includes AnPixJg2 (UniProtKB Q8YXY7 ) and NpR6012g4 (UniProtKB B2IU14 ) that have been extensively characterized. Here we examine the forward Pr photodynamics of NpR3784 (UniProtKB B2J457 ), a representative member of the second R/G CBCR subfamily. Using broadband transient absorption pump-probe spectroscopy, we characterize both primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) forward (Pr → Pg) photodynamics and compare the results to temperature-jump cryokinetics measurements. Our studies show that primary isomerization dynamics occur on an ∼10 ps timescale, yet remarkably, the red-shifted primary Lumi-Rf photoproduct found in all photoactive canonical R/G CBCRs examined to date is extremely short-lived in NpR3784. These results demonstrate that differences in reaction pathways reflect the evolutionary history of R/G CBCRs despite the convergent evolution of their photocycle end products.

Reverse Photodynamics of the Noncanonical Red/Green NpR3784 Cyanobacteriochrome from Nostoc punctiforme.

In the companion paper (10.1021/acs.biochem.8b01274), we examined the forward Pr photodynamics of NpR3784 (UniProtKB B2J457 ), a representative member of a noncanonical red/green (R/G) cyanobacteriochrome (CBCR) subfamily. Here the reverse Pg → Pr photodynamics of NpR3784 was studied by broadband transient absorption pump-probe spectroscopy. Primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) photodynamics were characterized over nine decades of time, which also were complemented with temperature-jump cryokinetics measurements. In contrast with canonical R/G CBCRs, the NpR3784 reverse photoconversion yielded two spectrally distinct primary photoproducts, Lumi-Go and Lumi-Gr, which decay on different time scales. The two primary photoproducts of NpR3784 equilibrate on the 40 ns time scale and subsequently propagate as a single intermediate population into Pr. Such heterogeneity could arise from differences in the direction of D-ring rotation, in chromophore protonation or hydrogen bonding, or in the mobility of protein residues or of solvent water nearby the chromophore or some combination therein. We conclude that the atypical photodynamics of NpR3784 reflects chromophore-protein interactions that differ from those present in the canonical R/G CBCR family.

Conservation and diversity in the secondary forward photodynamics of red/green cyanobacteriochromes.

Cyanobacteriochromes (CBCRs) are photosensitive proteins that are distantly related to the phytochrome family of photoreceptors and, like phytochromes, exhibit photoactivity initiated by the excited-state photoisomerization of a covalently bound bilin chromophore. The canonical red/green photoswitching sub-family is the most studied class of CBCRs studied to date. Recently, a comparative study of the ultrafast (100 fs-10 ns) forward photodynamics of nine red/green photoswitching CBCR domains isolated from Nostoc punctiforme were reported (S. M. Gottlieb, P. W. Kim, C.-W. Chang, S. J. Hanke, R. J. Hayer, N. C. Rockwell, S. S. Martin, J. C. Lagarias and D. S. Larsen, Conservation and Diversity in the Primary Forward Photodynamics of Red/Green Cyanobacteriochromes, Biochemistry, 2015, 54, 1028-1042). We extend this study by characterizing the secondary (10 ns-1 ms) forward photodynamics of eight red/green photoswitching CBCRs from N. punctiforme with broadband time-resolved absorption spectroscopy. We demonstrate that the dynamics of these representative red/green CBCRs can be separated into two coexisting pathways involving a photoactive pathway that is successful in generating the terminal light-adapted 15EPg population and an unsuccessful pathway that stalls after generating a meta-stable Lumi-Of intermediate. The photoactive pathway evolves through a similar mechanism from excitation of the dark-adapted 15ZPr state to generate a far-red absorbing Lumi-Rf and then via a succession of blue-shifting photointermediates to ultimately generate the 15EPg state. This suggests a steady deviation from planarity of the bilin chromophore during the dynamics. While, the general mechanism for this evolution is conserved among these CBCBs, the timescales of these dynamics deviate significantly. Only half of the characterized CBCRs exhibit the unproductive pathways due to photoexcitation of dark-adapted 15ZPo sub-population that upon photoexcitation generates a meta-stable Lumi-Of intermediate, which eventually decays back to the 15ZPo subpopulation. 15ZPo is ascribed the horizontal Asp657 configuration that disrupts H-bonding with the chromophore in the dark-adapted state; its presence can be identified via enhanced absorption of high-energy tail of the electronic absorption spectrum.