Stereoselective Intramolecular [2+2] Trapping of 1,2-Cyclohexadienes: a Route to Rigid, Angularly Fused Tricyclic Scaffolds.

1,2-Cyclohexadienes generated under mild fluoride-mediated desilylative conditions undergo efficient intramolecular [2+2] trapping, providing tricyclic alkylidene cyclobutanes with complete diastereoselectivity for the cis-fused products. Pendent styrenes or electron-deficient olefins can trap simple 1,2-cyclohexadienes or their oxygenated counterparts, with 14 substrates being disclosed. Reactions proceed at ambient temperature using just cesium fluoride in up to 91 % yield, and the necessary precursors are easily accessed from substituted 2-bromocyclohexenones. Multiple synthetic routes have been developed to install the appropriate functional groups required for [2+2] trapping.

A Novel Dialkylamino-Functionalized Chalcone, DML6, Inhibits Cervical Cancer Cell Proliferation, In Vitro, via Induction of Oxidative Stress, Intrinsic Apoptosis and Mitotic Catastrophe.

In this study, we designed, synthesized and evaluated, in vitro, novel chalcone analogs containing dialkylamino pharmacophores in the cervical cancer cell line, OV2008. The compound, DML6 was selective and significantly decreased the proliferation of OV2008 and HeLa cells in sub-micromolar concentrations, compared to prostate, lung, colon, breast or human embryonic kidney cell line (HEK293). DML6, at 5 µM, arrested the OV2008 cells in the G2 phase. Furthermore, DML6, at 5 µM, increased the levels of reactive oxygen species and induced a collapse in the mitochondrial membrane potential, compared to OV2008 cells incubated with a vehicle. DML6, at 5 µM, induced intrinsic apoptosis by significantly (1) increasing the levels of the pro-apoptotic proteins, Bak and Bax, and (2) decreasing the levels of l the anti-apoptotic protein, Bcl-2, compared to cell incubated with a vehicle. Furthermore, DML6, at 5 and 20 µM, induced the cleavage of caspase-9, followed by subsequent cleavage of the executioner caspases, caspase-3 and caspase-7, which produced OV2008 cell death. Overall, our data suggest that DML6 is an apoptosis-inducing compound that should undergo further evaluation as a potential treatment for cervical cancer.

The Safety and Feasibility of Exoskeletal-Assisted Walking in Acute Rehabilitation After Spinal Cord Injury.

OBJECTIVE: To assess safety and feasibility for persons with acute spinal cord injury (SCI) using the robotic exoskeleton. DESIGN: Case series observational study. SETTING: A level-1 trauma center in Canada with both acute and tertiary inpatient SCI rehabilitation units. PARTICIPANTS: Eight male and 3 female (N=11) participants were recruited with a mean age of 41 years and with neurologic level of injury (C6-L2) and severity (American Spinal Injury Association Impairment Scale [AIS] A-D). The time since injury is a range of 3-15 weeks at the onset of training. INTERVENTIONS: Up to 25 one-hour sessions of exoskeletal-assisted walking gait training, with participants less than 6 months from initial SCI. MAIN OUTCOME MEASURES: Cardiopulmonary outcomes including blood pressure, heart rate, and peripheral oxygen saturation; and perceived physical exertion using the Borg CR10 Scale were recorded. Gait parameters were measured by 6-minute walk test (6MWT) and 10-meter walk test (10MWT). Up Time, walk time, and number of steps were detailed longitudinally. Safety was assessed with regard to pain, falls, and skin integrity. RESULTS: No serious adverse events occurred. Blood pressure decreased following initial sit to stand and increased during walking. Symptoms of hypotension were rare and improved with increased number of sessions. Perceived exertion was reported on average to be moderate (mean of 3.1). There was no significant increase in pain scores by Visual Analog Scale. On 6MWT, participants covered more distance (mean [m] ± SD, 117.1±11.7) in session 25 compared to session 2 (mean [m] ± SD, 47.6±6.6). On the 10MWT, all participants showed consistently improved gait speed; with participants traveling an average of 3.2 times faster during their last training session (mean [m/s] ± SD, 0.40±0.04) in comparison to session 2 (mean [m/s] ± SD, 0.12±0.01). CONCLUSIONS: Exoskeletal-assisted walking in acute rehabilitation (<6mo) following SCI appears to be both safe and feasible.

Neural, biomechanical, and physiological factors involved in sex-related differences in the maximal rate of isometric torque development.

OBJECTIVE: Recent research has reported that lower maximal rate of torque development (dτ/dt max) exhibited by females, relative to males, during knee extension can be accounted for by normalization to a maximal voluntary contraction (MVC); however, this was not seen in the upper limb. PURPOSE: The aim of the current work was to examine the contribution of maximum strength (τmax), twitch contraction time (CT), muscle fiber condition velocity (MFCV), and rate of muscle activation (Q30) to sex-differences in the dτ/dt max during maximal isometric dorsiflexion. METHODS: Thirty-eight participants (20 males; 18 females) performed both maximal voluntary and evoked isometric contractions of the tibialis anterior across 3 days. Ten maximal compound muscle action potentials were elicited and subsequently followed by three, 5-s contractions. From the recordings, MFCV, dτ/dt max, τmax, CT, electromechanical delay (EMD), root-mean squared (RMS) amplitude, peak-to-peak voltage (Vpp), and Q30 were calculated. RESULTS: An ANCOVA showed that τmax accounted for all the sex-differences in dτ/dt max (p = 0.96). There were no significant differences between groups with respect to MFCV, RMS amplitude, Vpp amplitude, or CT. However, there was a significant sex-difference in dτ/dt max, τmax, and Q30. Females had longer evoked EMD times compared with males (15.69 ± 10.57 ms versus 9.95 ± 3.46 ms; p = 0.01), but the voluntary EMD times were not different. CONCLUSION: The current research supports the work by Hannah et al. Exp Physiol 97:618-629, (2012) that normalization to MVC in the quadriceps is able to account for all sex-differences in rate of toque development in the lower limb.

qPCR-based phytoplankton abundance and chlorophyll a: A multi-year study in twelve large freshwater rivers across the United States.

Phytoplankton overgrowth, which characterizes the eutrophication or trophic status of surface water bodies, threatens ecosystems and public health. Quantitative polymerase chain reaction (qPCR) is promising for assessing the abundance and community composition of phytoplankton. However, applications of qPCR to indicate eutrophication and trophic status, especially in lotic systems, have yet to be comprehensively evaluated. For the first time, this study correlates qPCR-based phytoplankton abundance with chlorophyll a (the most widely used indicator of eutrophication and trophic status) in multiple freshwater rivers. From early summer to late fall in 2017, 2018, and 2019, we evaluated phytoplankton, chlorophyll a, pheophytin a, and the Trophic Level Index (TLI) in twelve large freshwater rivers in three regions (western, midcontinent, and eastern) in the United States. Chlorophyll a concentration had positive allometric correlations with qPCR-based phytoplankton abundance (adjusted R2 = 0.5437, p-value < 0.001), pheophytin a concentration (adjusted R2 = 0.3378, p-value <0.001), and TLI (adjusted R2 = 0.4789, p-value < 0.001). Thus, a greater phytoplankton abundance suggests a higher trophic status. This work also presents the numerical values of qPCR-based phytoplankton abundance defining the boundaries among trophic statuses (e.g., oligotrophic, mesotrophic, and eutrophic) of freshwater rivers. The sampling sites in the midcontinent rivers were more eutrophic because they had significantly higher chlorophyll a concentrations, pheophytin a concentrations, and TLI values than the sites in the western and eastern rivers. The higher phytoplankton abundance at the midcontinent sites confirmed their higher trophic status. By linking qPCR-based phytoplankton abundance to chlorophyll a, this study demonstrates that qPCR is a promising avenue to investigate the population dynamics of phytoplankton and the trophic status (or eutrophication) of freshwater rivers.

Spatial and Temporal Variability of Saxitoxin-Producing Cyanobacteria in U.S. Urban Lakes.

Harmful cyanobacterial blooms (HCBs) are of growing global concern due to their production of toxic compounds, which threaten ecosystems and human health. Saxitoxins (STXs), commonly known as paralytic shellfish poison, are a neurotoxic alkaloid produced by some cyanobacteria. Although many field studies indicate a widespread distribution of STX, it is understudied relative to other cyanotoxins such as microcystins (MCs). In this study, we assessed eleven U.S. urban lakes using qPCR, sxtA gene-targeting sequencing, and 16S rRNA gene sequencing to understand the spatio-temporal variations in cyanobacteria and their potential role in STX production. During the blooms, qPCR analysis confirmed the presence of the STX-encoding gene sxtA at all lakes. In particular, the abundance of the sxtA gene had a strong positive correlation with STX concentrations in Big 11 Lake in Kansas City, which was also the site with the highest quantified STX concentration. Sequencing analysis revealed that potential STX producers, such as Aphanizomenon, Dolichospermum, and Raphidiopsis, were present. Further analysis targeting amplicons of the sxtA gene identified that Aphanizomenon and/or Dolichospermum are the primary STX producer, showing a significant correlation with sxtA gene abundances and STX concentrations. In addition, Aphanizomenon was associated with environmental factors, such as conductivity, sulfate, and orthophosphate, whereas Dolichospermum was correlated with temperature and pH. Overall, the results herein enhance our understanding of the STX-producing cyanobacteria and aid in developing strategies to control HCBs.

Periodic Addition of Glucose Suppressed Cyanobacterial Abundance in Additive Lake Water Samples during the Entire Bloom Season.

Previously, we showed that prophylactic addition of glucose to Harsha Lake water samples could inhibit cyanobacteria growth, at least for a short period of time. The current study tested cyanobacterial control with glucose for the entire Harsha Lake bloom season. Water samples (1000 ml) were collected weekly from Harsha Lake during the algal-bloom season starting June 9 and lasting until August 24, 2022. To each of two 7-liter polypropylene containers, 500 ml of Harsha Lake water was added, and the containers were placed in a controlled environment chamber. To one container labeled "Treated," 0.15 g of glucose was added, and nothing was added to the container labeled "Control." After that, three 25 ml samples from each container were collected and used for 16S rRNA gene sequencing each week. Then 1000 ml of Harsha Lake water was newly collected each week, with 500 ml added to each container, along with the addition of 0.15 g glucose to the "Treated" container. Sequencing data were used to examine differences in the composition of bacterial communities between Treated and Control containers. Treatment with glucose altered the microbial communities by 1) reducing taxonomic diversity, 2) largely eliminating cyanobacterial taxa, and 3) increasing the relative abundance of subsets of non-cyanobacterial taxa (such as Proteobacteria and Actinobacteriota). These effects were observed across time despite weekly inputs derived directly from Lake water. The addition of glucose to a container receiving weekly additions of Lake water suppressed the cyanobacterial populations during the entire summer bloom season. The glucose appears to stimulate the diversity of certain bacterial taxa at the expense of the cyanobacteria.

Using cyanobacteria and other phytoplankton to assess trophic conditions: A qPCR-based, multi-year study in twelve large rivers across the United States.

Phytoplankton is the essential primary producer in fresh surface water ecosystems. However, excessive phytoplankton growth due to eutrophication significantly threatens ecologic, economic, and public health. Therefore, phytoplankton identification and quantification are essential to understanding the productivity and health of freshwater ecosystems as well as the impacts of phytoplankton overgrowth (such as Cyanobacterial blooms) on public health. Microscopy is the gold standard for phytoplankton assessment but is time-consuming, has low throughput, and requires rich experience in phytoplankton morphology. Quantitative polymerase chain reaction (qPCR) is accurate and straightforward with high throughput. In addition, qPCR does not require expertise in phytoplankton morphology. Therefore, qPCR can be a useful alternative for molecular identification and enumeration of phytoplankton. Nonetheless, a comprehensive study is missing which evaluates and compares the feasibility of using qPCR and microscopy to assess phytoplankton in fresh water. This study 1) compared the performance of qPCR and microscopy in identifying and quantifying phytoplankton and 2) evaluated qPCR as a molecular tool to assess phytoplankton and indicate eutrophication. We assessed phytoplankton using both qPCR and microscopy in twelve large freshwater rivers across the United States from early summer to late fall in 2017, 2018, and 2019. qPCR- and microscope-based phytoplankton abundance had a significant positive linear correlation (adjusted R2 = 0.836, p-value < 0.001). Phytoplankton abundance had limited temporal variation within each sampling season and over the three years studied. The sampling sites in the midcontinent rivers had higher phytoplankton abundance than those in the eastern and western rivers. For instance, the concentration (geometric mean) of Bacillariophyta, Cyanobacteria, Chlorophyta, and Dinoflagellates at the sampling sites in the midcontinent rivers was approximately three times that at the sampling sites in the western rivers and approximately 18 times that at the sampling sites in the eastern rivers. Welch's analysis of variance indicates that phytoplankton abundance at the sampling sites in the midcontinent rivers was significantly higher than that at the sampling sites in the eastern rivers (p-value = 0.013) but was comparable to that at the sampling sites in the western rivers (p-value = 0.095). The higher phytoplankton abundance at the sampling sites in the midcontinent rivers was presumably because these rivers were more eutrophic. Indeed, low phytoplankton abundance occurred in oligotrophic or low trophic sites, whereas eutrophic sites had greater phytoplankton abundance. This study demonstrates that qPCR-based phytoplankton abundance can be a useful numerical indicator of the trophic conditions and water quality in freshwater rivers.

Genomic Characterization and Wetland Occurrence of a Novel Campylobacter Isolate from Canada Geese.

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland-a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

Reliability of a simple method for determining muscle fiber conduction velocity.

INTRODUCTION: This study evaluated the reliability of muscle fiber conduction velocity (MFCV) measurement. METHODS: Forty healthy, young participants performed isometric dorsiflexion of the foot on 3 non-consecutive days. The reliability of force, root-mean-square (RMS) amplitude of the surface electromyographic (sEMG) signal, and MFCV were evaluated using the intraclass correlational analysis of variance technique. RESULTS: The means across test days for all measures exhibited slight changes (<5%) and were considered stable. All measures exhibited remarkable consistency within subjects as indicated by high intraclass correlation coefficients (0.83-0.98). CONCLUSIONS: The procedures resulted in highly reliable MFCV values, and included: (1) electric identification of motor points prior to electrode placement; (2) twitch identification of muscle fiber orientation to guide initial electrode placement; (3) rotation of electrodes clockwise or counter-clockwise to maximize the similarity and delay of compound muscle action potentials across all detection surfaces; and (4) minimization of synergistic activity during voluntary contractions.

Exoskeleton use in acute rehabilitation post spinal cord injury: A qualitative study exploring patients' experiences.

CONTEXT/OBJECTIVE: Spinal cord injury (SCI) is intensely life altering, affecting multiple body systems and functions, including the ability to walk. Exoskeleton assisted walking (EAW) is a rehabilitation tool that aims to support locomotor training, yet little is known about the patient experience. The purpose of this qualitative study, part of a prospective observational case series, was to increase our understanding of SCI patient experience using a robotic exoskeleton in this acute post-injury period. DESIGN: A qualitative descriptive approach was implemented in this study, with the aim to explore and understand participants' experience with EAW training. PARTICIPANTS/SETTING: Nine of the 11 participants enrolled in the observational study agreed to participate in an interview. All participants had suffered a SCI, and had received their trauma care and inpatient rehabilitation at a tertiary center in Calgary, Alberta, Canada. RESULTS: The benefits to EAW use described by participants were primarily psychological and included the joy of eye level contact, excitement at being able to walk with assistance, improvement in mood, and hope for the future. Potential physiological benefits include increased strength, decreased spasticity and reduced pain. Challenges to EAW use include weakness and fatigue, and a fear of incontinence. CONCLUSION: Qualitative research will continue to be an important component in future research on the use of EAW training as part of the rehabilitation process. Increasing understanding of the participants experience with this novel therapeutic modality and technology will be fundamental to improve its implementation in clinical practice.

Oral and dermal toxicity of alkenones extracted from Isochrysis species.

Isochrysis is commercially available marine algae used for animal feed, human nutrient supplements, and biodiesel. The Isochrysis species is one of five genera of haptophytes that produces unique, long-chain lipids known as alkenones that are promising new ingredients for green cosmetics, personal care products and pharmaceutical delivery. However, there is a lack of toxicity data for alkenones in animals, thus limiting their use in humans. In this study, we performed acute oral, acute dermal, and repeated 28-day dermal toxicity studies, using female SAS Sprague Dawley Rats. Our behavioral studies indicated that the specific alkenones had no overt behavioural effects at oral doses up to 4000 mg/kg. In the acute and chronic dermal toxicity studies, the alkenones produced less irritation and did not significantly damage the skin based on the Draize skin reaction scale and trans-epidermal water loss readings compared to the positive control, 1% sodium lauryl sulfate. Overall, our results indicated that alkenones are safe in Sprague Dawley rats, suggesting that they could be used for both oral and dermal formulations, although additional studies will be required.

Strain-Activated Diels-Alder Trapping of 1,2-Cyclohexadienes: Intramolecular Capture by Pendent Furans.

Intramolecular [4 + 2] cycloaddition reactions of substituted 1,2-cyclohexadienes with pendent furans enables the synthesis of complex tetracyclic scaffolds in a single step under mild conditions. All Diels-Alder cycloadducts were obtained as single diastereomers, assigned as the endo isomer. Substrates were easily assembled via Stork-Danheiser alkylation of 3-ethoxy-2-bromocyclohex-2-enone to accommodate a range of tethers and furan traps. Cleavage of enol acetate moieties resulted in room-temperature Diels-Alder cycloreversion to tethered furyl cyclohexenones.

2,3-Diaryl-3H-imidazo[4,5-b]pyridine derivatives as potential anticancer and anti-inflammatory agents.

In this study we examined the suitability of the 3H-imidazo[4,5-b]pyridine ring system in developing novel anticancer and anti-inflammatory agents incorporating a diaryl pharmacophore. Eight 2,3-diaryl-3H-imidazo[4,5-b]pyridine derivatives retrieved from our in-house database were evaluated for their cytotoxic activity against nine cancer cell lines. The results indicated that the compounds showed moderate cytotoxic activity against MCF-7, MDA-MB-468, K562 and SaOS2 cells, with K562 being the most sensitive among the four cancer cell lines. The eight 2,3-diaryl-3H-imidazo[4,5-b]pyridine derivatives were also evaluated for their COX-1 and COX-2 inhibitory activity in vitro. The results showed that compound 3f exhibited 2-fold selectivity with IC50 values of 9.2 and 21.8 µmol/L against COX-2 and COX-1, respectively. Molecular docking studies on the most active compound 3f revealed a binding mode similar to that of celecoxib in the active site of the COX-2 enzyme.

Tackling multidrug resistance mediated by efflux transporters in tumor-initiating cells.

INTRODUCTION: Expression of the multifunctional ATP-binding cassette (ABC) efflux transporter gene family is a well-established mechanism for protecting cancer stem cells (CSCs) from damage or death due to toxins. The outcome of such protection makes CSCs innately multidrug resistant (MDR) to conventional chemotherapy. AREAS COVERED: While research has focused on gaining better insight into the role of ABC transporters in CSC drug resistance, various strategies to circumvent the function of these transporters have been proposed, including inhibition of transporter function through targeted tyrosine kinase inhibitors, competitive and allosteric modulators, shRNA-mediated inhibition, nanoparticle-mediated delivery of inhibitors, and modulating the regulation of transcriptional and signaling pathways involving ABC transporters. This review highlights the role of MDR mediated by ABC transporters, particularly in CSCs, and the current progress and pitfalls of strategies to circumvent MDR in CSCs. EXPERT OPINION: Cancer stem cells are now a subject of intense research, as it is hypothesized that these progenitors predominantly beget tumorigenesis, chemoresistance, and metastasis. Consequently, the design and synthesis of more effective ABC transporter inhibitors, to increase cytotoxic drug concentrations in CSCs (thus increasing their eradication), is a promising approach for the field of oncology.

Estimation of impulse response between electromyogram signals for use in conduction delay distribution estimation.

The time delay between two surface electromyograms (EMGs) acquired along the conduction path is used to estimate mean action potential conduction velocity. Modeling the linear impulse response between "upstream" and "downstream" EMG signals permits an estimate of the distribution of velocities, providing more information. In this work, we analyzed EMG from bipolar electrodes placed on the tibialis anterior of 36 subjects, using an inter-electrode distance of 10 mm. Regularized least squares was used to fit the coefficients of a finite impulse response model. We trained the model on one recording, then tested on two others. The optimum correlation between the model-predicted and actual EMG averaged 0.70. We also compared estimation of the mean conduction delay from the peak time of the impulse response to the "gold standard" peak time of the cross-correlation between the upstream and downstream EMG signals. Optimal models differed from the gold standard by 0.02 ms, on average. Model performance was influenced by the regularization parameters. The impulse responses, however, incorrectly contained substantive power at very low time delays, causing delay distribution estimates to exhibit high probabilities at very short conduction delays. Unrealistic distribution estimates resulted. Larger inter-electrode spacing may be required to alleviate this limitation.

Modeling the impulse response between pairs of EMG signals to estimate conduction delay distribution.

Mean electromyogram (EMG) conduction delay is often estimated as the average time delay between two surface EMG recordings arranged along the conduction path. It has previously been shown that the complete distribution of conduction delays can be estimated from the impulse response relating the "upstream" EMG recording to the "downstream" recording. In this work, we examined regularized least squares methods for estimating the impulse response, namely the pseudo-inverse with small singular values discarded and post hoc lowpass filtering. Performance was evaluated by training the model to one recording, then testing on others. Correlation between model-predicted EMG and measured EMG was assessed for 36 subjects, using EMG recordings with 5 mm inter-electrode spacing. The best correlation was 0.86, on average, for both regularization methods. We additionally compared the mean conduction delay computed from the "gold standard" cross-correlation method to the peak time of the impulse response. The best models differed by 0.01 ms, on average, for both regularization methods. Nonetheless, the impulse responses exhibited excessive energy near zero time, causing delay distribution estimates to exhibit high probabilities at unphysiological short time delays. Inter-electrode spacing larger than 5 mm may be required to alleviate this limitation.