Comparative study of production of dextransucrase and dextran by cells of Leuconostoc mesenteroides immobilized on Celite and in calcium alginate beads

In fed-batch fermentation, cells of L. mesenteroides immobilized on three types of Celite were used to produce dextransucrase (DS) followed by production of dextran. A layer of calcium alginate on the porous Celite R630 particles improved their mechanical stability, increased the amount of soluble DS produced and decreased the cell leakage from the highly porous support. Enzyme production with the immobilized cell cultures was significantly affected by both pore and particle size. Immobilized cultures using Celite R648 (average particle radius of 200 mum and pore size of 0.14 mum) produced the highest total enzymatic activity, followed by Celite R633, alginate-coated Celite R630, Celite R630, and then calcium alginate beads. Culture of free cells produced about 18% more total enzymatic activity than immobilized cells in calcium alginate beads, but about 64% less than immobilized cells on Celite R630. It is expected that larger amounts of enzymatic activity than measured are immobilized inside the alginate-coated Celite R630 and calcium alginate beads due to the mass transfer limitation conferred by the dextran product formed therein. The dextran yield from conversion of sucrose to dextran and fructose with all such enzyme-enriched, immobilized-cell cultures was higher than that obtained from free-cell culture under similar conditions.     

Phase-separated protein droplets of amyotrophic lateral sclerosis-associated p62/SQSTM1 mutants show reduced inner fluidity

Several amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43, and hnRNPA1 demonstrate liquid–liquid phase separation, and their disease-related mutations correlate with a transition of their liquid droplet form into aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD), and Paget’s disease of bone. SQSTM1/p62 protein forms liquid droplets through interaction with ubiquitinated proteins, and these droplets serve as a platform for autophagosome formation and the antioxidative stress response via the LC3-interacting region (LIR) and KEAP1-interacting region (KIR) of p62, respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in the LIR and KIR disrupt liquid droplet formation leading to defects in autophagy, the stress response, or both. To evaluate the effects of ALS/FTD-related p62 mutations in the LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway, as well as on autophagic turnover, we developed systems to monitor each of these with high sensitivity. These methods such as intracellular protein–protein interaction assay, doxycycline-inducible gene expression system, and gene expression into primary cultured cells with recombinant adenovirus revealed that some mutants, but not all, caused reduced NRF2 activation and delayed autophagic cargo turnover. In contrast, while all p62 mutants demonstrated sufficient ability to form liquid droplets, all of these droplets also exhibited reduced inner fluidity. These results indicate that like other ALS-related mutant proteins, p62 missense mutations result in a primary defect in ALS/FTD via a qualitative change in p62 liquid droplet fluidity.     

Inclusion: an individual pursuit requiring organization-level investment to sustain it

If this was not happening in the midst of the COVID-19 pandemic, I imagine that I would be speaking these words instead of writing them on my laptop. Even so, I am so jazzed for this opportunity! No word or phrase describes what I am feeling in this moment in receiving the 2021 American Society for Cell Biology Prize for Excellence in Inclusivity. It is certainly an honor to be recognized in this way. I am grateful to the Howard Hughes Medical Institute for awarding me additional resources to keep on keeping on. My approach to finding the connection between people and their science certainly could use the monetary support. Resources open doors. At the same time that I am grateful for the attention, I am not exactly sure what to do with the spotlight. Importantly, there are a host of other folks out there also doing amazing things who have never been recognized. Let’s work to ensure that their contributions are supported, appreciated, and recognized. Instead of focusing the spotlight on me, I would rather redirect it to recognize my foundational influences. I also hope to encourage the need for institutional approaches beyond celebrating individual accomplishment.

O. A. Quintero‐CarmonaJo Rae Wright was my graduate advisor and the model for how I have tried to work with my students and colleagues to support their opportunities while also “doing science.” I wanted to start graduate school as soon as I could after graduating college, so after letting the Cell and Molecular Biology Program at Duke University know that I was accepting their offer, I started thumbing through their program booklet looking for labs with interesting research projects (a web presence wasn’t even really a thing for departments in 1996). I worked alphabetically and contacted a handful of labs one at a time to see whether anyone was willing to take on an early-rotation student. It was an unusual request for the way that the program had operated previously, and Jo Rae was the only person to agree to it. I don’t remember exactly, but she said something like, “We accepted you into the program, so I would be happy to host your first rotation.” The sense that I got was that, within the limits of her time and resources, she was willing to become my mentor because I needed one. She trusted the admissions process, so why not bring an eager student into the lab. I spent the summer settling in to the life of a graduate student—sort of.At first, I was bad at graduate school. I am curious about all sorts of things, which means I am also easily pulled in too many directions. In that first year of school I spent way too much time simply visiting other students in my cohort to see what it was that they were up to each day. I cannot imagine how distracting I must have been to them and probably extremely irritating to their PIs as well. If you were in Cell Biology at Duke in 1996–97, I am sincerely grateful that you tolerated my shenanigans. Where others might have taken me to task, Jo Rae looked for opportunities to redirect my energies more productively. She and another professor, Dan Kiehart, guided me toward participating in the Physiology Course at the Marine Biological Laboratory, where I learned what I needed to do to be a scientist in a way that would not have been possible otherwise. While there, I saw PIs working with students chasing the joy of discovery, and it felt like it was purely for the sake of a deeper understanding of biology and preparing the next generation of scientists to do the same. Resources gave us the liberty to focus on scientific discovery with minimal concern for where would be the highest profile place to publish. Although I acknowledge that the summer course environments may not be the most representative of the daily life of a scientist at a home institution, such an opportunity left a mark—I wanted to come as close as I could to emulating that environment when I got back to Duke and (eventually) when I had the chance to run a research group and teach students.Along the way, Jo Rae made sure to include me and my fellow lab mates in all aspects of the science. At national meetings she included us at every step, introducing us to her contemporaries and putting us in spaces where we would rub elbows with luminaries in the field. When we were in those environments, she made sure that I felt like a junior colleague. I cannot recall ever feeling like a “trainee.” Back home at Duke, I had opportunities to do everything that a scientist might do in addition to “sciencing.” Sure, I would write papers, contribute to grants, and be part of her review of papers. I was also encouraged to mentor undergraduates, teach, advocate for federal funding at the time of the National Institutes of Health (NIH) doubling, and plan events for Duke’s summer undergraduate research program, if I so chose. Similarly, when I expressed an interest in focusing on science with undergraduates, she was 100% on board with finding ways to combine my graduate school commitments with teaching and mentoring opportunities. Importantly, at a time when expressing interest in an “alternative career” was not always supported by faculty mentors, Jo Rae encouraged me to seek out only those potential postdoctoral mentors who would actively support my goals. Not only that, she went out of her way to find out what options I might have, which led to her learning about the NIH-funded Institutional Research and Academic Career Development Award postdoctoral programs in their first year of existence.In a sentence, because Jo Rae was 100% invested in including me in science by finding the framework that best suited my interests and potential, I grew into my success. This was a form of success that wasn’t decided by someone else; I had defined it for myself with Jo Rae serving as a true advisor in every sense of the word—she was in it for me. She helped to build the crucial foundations that helped me find the opportunities that matched my goals. As a result of her influence, I have also had the strength to make some critical, nontraditional choices along the way. Her mentorship style was tailored to each individual’s needs. She invested the time to figure out our strengths, and also learned which levers would motivate us to meet our potential. The members of her lab became successes because she helped all of us to both define success and achieve our own version of it. Such a personal approach is extremely powerful. Jo Rae passed away in 2012, and with her passing I lost the most important influence in my professional life. Duke University and the pulmonary physiology community lost an example for inclusive mentorship and a significant amount of capacity for such an approach. Since her passing, multiple awards have been established to honor Jo Rae’s legacy as an outstanding woman in science. I would argue that mentoring of junior colleagues may be a more significant legacy than her scientific output. Jo Rae is deserving of this award.Recognitions such as this one are an important way to amplify examples of what we often say we hope to achieve as a department, an institution, or a scientific society. However, if our focus is solely on the efforts of individuals, we are missing an opportunity. While I am humbled to be considered in the same league as the previous award recipients, we are each in our own way scrambling to do what we can while we can do it. When individuals have some positive outcomes, our institutions and organizations will celebrate what these folks have done as they have played some role in supporting these opportunities. Although what we do is worthwhile, it is really hard to do it successfully and sustainably without proper institutional support. We each face hinderances that can undermine the work that we want to take on. Burnout is a real outcome of doing the work that we care about and that our organizations publicly state is important. This is especially true in environments where that work is undervalued and underresourced. You do not have to do a very extensive internet search to identify where the institutions that have supported my work also have exclusionary legacies and current negative influences that continue to hinder their potential for broader, more meaningful progress. In many instances inclusion has yet to be baked into institutional culture in a way that impacts how organizations operate. Although I have had some institutional support to develop a career modeled on what I experienced under Jo Rae’s mentorship, the students and faculty at these institutions know that what gets headlines can often be an exceptional situation, rather than a typical everyday experience. Rather than showcasing the good work of individuals in their ranks, an organization should devote itself to furthering the idea that it is willing to make significant institutional investments in that good work. By building the internal infrastructure and capacity to support inclusion efforts, organizations would demonstrate that inclusion is an essential component of the institutional standard practice. The positive outcomes that this award is intended to highlight would then be a shared characteristic of the community. A shared vision paired with shared effort and resource-support might cut down on burnout of those currently carrying more than their share of the load.I imagine that the idea for these awards is to celebrate good work while also demonstrating to other individuals what is possible. With that in mind, if institutions worked at using the example of those in the vanguard as a way to build structures that value and support inclusive approaches, they would increase their own ability to serve their constituents. They may also influence other institutions to do the same. My graduate institution benefited from Jo Rae’s work while she was present and was beginning to institutionalize her view of inclusion in the last years of her life. As Dean of the Graduate School, the model for how she ran her lab informed her vision for graduate education campus-wide. She wanted to build a structure that would identify, recruit, and retain talent. She wanted to provide that talent with opportunities to become expert in how they wanted to contribute to the world. By ensuring that they had access to the relevant experiences and skills, she hoped to support them as they set themselves up for success as they defined it.I accept this award in honor of Jo Rae Wright, and on behalf of the students who have trusted me. All I have ever wanted was to be able to recreate for my undergraduates what Jo Rae had done for the people under her wing. I am building a career around that goal as part of a department keenly supportive of these efforts. My hope is that other individuals will develop their own approaches to inclusion because they find themselves in supportive institutional environments. More importantly, I would like to see organizations begin to truly prioritize inclusive approaches through funding and through policy. Institutions could make sufficient resources available to support inclusive efforts and allow creativity in how faculty mobilize those resources. Just as Jo Rae had the flexibility to adjust to our needs, institutional efforts will benefit when limited resource access is not a hindrance to inclusive excellence. Additionally, it will be critical to acknowledge the time and effort that such endeavors require in evaluating faculty contributions. It can no longer be the icing on the cake of a portfolio—developing inclusive capacity has to be recognized as an essential component of our work. Until these changes take root at the institutional level, this kind of work may shine brightly, but will continue to be stochastic and short-lived. All those efforts “will be lost in time, like tears in rain.” It is on all of us to prevent such a tragic ending.     

The effect of post-lunch napping on mood,reaction time,and antioxidant defense during repeated sprint exercice

To compare the effects of two nap opportunities (20 and 90 min) to countermeasure the transient naturally occurring increased sleepiness and decreased performances during the post-lunch dip (PLD). Fourteen highly trained judokas completed in a counterbalanced and randomized order three test sessions (control (No-nap), 20- (N20) and 90-min (N90) nap opportunities). Test sessions consisted of the running-based anaerobic sprint test (RAST), simple and multiple-choice reaction times (MCRT) and the Epworth sleepiness scale (ESS). From the RAST, the maximum (P_max), mean (P_mean) and minimum (P_min) powers were calculated. Blood samples were taken before and after the RAST to measure the effect of pre-exercise napping on energetic and muscle damage biomarkers and antioxidant defense. N20 increased P_max and P_mean compared to No-nap (p < 0.001, d = 0.59; d = 0.66) and N90 (p < 0.001, d = 0.98; d = 0.72), respectively. Besides, plasma lactate and creatinine increased only when the exercise was performed after N20. Both N20 (p < 0.001, d = 1.18) and N90 (p < 0.01, d = 0.78) enhanced post-exercise superoxide dismutase activity compared to No-nap. However, only N20 enhanced post-exercise glutathione peroxidase activity (p < 0.001, d = 1.01) compared to pre-nap. Further, MCRT performance was higher after N20 compared to No-nap and N90 (p < 0.001, d = 1.15; d = 0.81, respectively). Subjective sleepiness was lower after N20 compared to No-nap (p < 0.05, d = 0.92) and N90 (p < 0.01, d = 0.89). The opportunity to nap for 20 min in the PLD enhanced RAST, MCRT performances, and antioxidant defense, and decreased sleepiness. However, the opportunity of 90 min nap was associated with decreased repeated sprint performances and increased sleepiness, probably because of the sleep inertia.     

Study on the interaction between erythrosine B and the cardiac drug amiodarone using fluorescence,scattering, and absorbance spectra and their analytical application

In an acidic buffered solution, erythrosine B can react with amiodarone to form an association complex, which not only generates great enhancement in resonance Rayleigh scattering (RRS) spectrum of erythrosine B at 346.5 nm but also results in quenching of fluorescence spectra of erythrosine B at λ_emission = 550.4 nm/λ_excitation = 528.5 nm. In addition, the formed erythrosine B–amiodarone complex produces a new absorbance peak at 555 nm. The spectral characteristics of the RRS, absorbance, and fluorescence spectra, as well as the optimum analytical conditions, were studied and investigated. As a result, new spectroscopic methods were developed to determine amiodarone by utilizing erythrosine B as a probe. Moreover, the ICH guidelines were used to validate the developed RRS, photometric, and fluorimetric methods. The enhancements in the absorbance and the RRS intensity and the decrease in the fluorescence intensity of the used probe were proportional to the concentration of amiodarone in ranges of 2.5–20.0, 0.2–2.5, and 0.25–1.75 μg/mL, respectively. Furthermore, limit of detection values were 0.52 ng/mL for the spectrophotometric method, 0.051 μg/mL for the RRS method, and 0.075 μg/mL for the fluorimetric method. Moreover, with good recoveries, the developed spectroscopic procedures were applied to analyze amiodarone in its commercial tablets.     

Curcumin,its derivatives,and their nanoformulations: Revolutionizing cancer treatment

Curcumin is a natural compound derived from turmeric and can target malignant tumor molecules involved in cancer propagation. It has potent antioxidant activity, but its effectiveness is limited due to poor absorption and rapid elimination from the body. Various curcumin derivatives have also shown anticancer potential in in-vitro and in-vivo models. Curcumin can target multiple signaling pathways involved in cancer development/progression or induce cancer cell death through apoptosis. In addition, curcumin and its derivatives could also enhance the effectiveness of conventional chemotherapy, radiation therapy and reduce their associated side effects. Lately, nanoparticle-based delivery systems are being developed/explored to overcome the challenges associated with curcumin's delivery, increasing its overall efficacy. The use of an imaging system to track these formulations could also give beneficial information about the bioavailability and distribution of the nano-curcumin complex. In conclusion, curcumin holds significant promise in the fight against cancer, especially in its nanoform, and could provide precise delivery to cancer cells without affecting normal healthy cells.     

Oviduct fluid and heparin induce similar surface changes in bovine sperm during capacitation: A flow cytometric study using lectins

Eight different lectins conjugated to fluorescein isothiocyanate (FITC) were used to screen for sperm plasma membrane changes during in vitro capacitation of bovine sperm. Analysis of lectin binding to sperm was done using flow cytometry. Of the eight lectins, only Triticum vulgaris (wheat germ agglutinin, WGA) binding to sperm was altered with capacitation. Capacitation of bovine sperm by heparin was found to decrease WGA binding to sperm by 78% (P < 0.05). The effect of capacitation by oviduct fluid was next compared with capacitation by heparin for changes in WGA binding to sperm. The effect of inhibiting capacitation with glucose on WGA binding was also determined. WGA-bound sperm were detected by flow cytometry as being present in two fluorescence peaks defined as low fluorescence (A) or high fluorescence (B) intensity. The percentage of sperm in peak A was greater for heparin and oviduct fluid-treated sperm compared to sperm incubated under noncapacitating conditions in only culture medium (P < 0.001). Capacitation with either heparin or oviduct fluid was inhibited by glucose as assessed by the ability of lysophosphatidylcholine (100 μg/ml) to induce acrosome reactions. Glucose also reduced the percentage of sperm in peak A for both heparin- and oviduct fluid-treated sperm (P < 0.01). We conclude that heparin or oviduct fluid induced changes on the sperm plasma membrane during capacitation. Binding sites for WGA on sperm were either structurally altered or lost during capacitation. © 1996 Wiley-Liss, Inc.     

Responses of Tropical Understory Plants to a Severe Drought: Tolerance and Avoidance of Water Stress1

Shade-tolerant understory shrubs and subcanopy trees constitute most of the woody species in Neotropical moist forest, but studies demonstrating physiological differences among these species are few. Shade-tolerant species that coexist in the forest understory exhibit differences in leaf life span that have been associated with variation in physiological traits. We hypothesized that water relations of understory species with widely divergent leaf life spans differ in response to drought. Although severe drought is infrequent in Neotropical moist forest, we studied the water relations of shade-tolerant understory species with short or long leaf life spans during the severe 1991-1992 dry season on Barro Colorado Island, Panama. The predawn leaf water potential declined to -2.8 and -3.6 MPa during the dry season in Hybanthus prunifolius and Psychotria horizontalis, respectively, two species with short leaf life spans, but remained above -1.3 MPa in two species with long leaf life spans, Swartzia simplex and Ouratea lucens. The midday leaf water potential dropped as low as -3.4 and -4.5 MPa for H. prunifolius and P. horizontalis, respectively. The osmotic potential of H. prunifolius and P. horizontalis and another species with short leaf life span, Alms blackiana, decreased early in the dry season, a period during which all three had substantially negative predawn water potential. In contrast, the osmotic potential of S. Simplex, O. lucens, and Licania platypus, a third species with long leaf life span, declined late in the dry season, even though we observed little change in predawn water potential for S. simplex and O. lucens. We conclude that the variable and potentially severe dry season in Neotropical moist forest can be sufficiently intense to severely limit soil moisture availability for understory plants. H. prunifolius and P. horizontalis tolerated dehydration, whereas S. simplex and O. lucens postponed dehydration.     

Evaluation of Microbiome Alterations Following Consumption of BIOHM,a Novel Probiotic

Gastrointestinal microbiome dysbiosis may result in harmful effects on the host, including those caused by inflammatory bowel diseases (IBD). The novel probiotic BIOHM, consisting of Bifidobacterium breve, Saccharomyces boulardii, Lactobacillus acidophilus, L. rhamnosus, and amylase, was developed to rebalance the bacterial–fungal gut microbiome, with the goal of reducing inflammation and maintaining a healthy gut population. To test the effect of BIOHM on human subjects, we enrolled a cohort of 49 volunteers in collaboration with the Fermentation Festival group (Santa Barbara, CA, USA). The profiles of gut bacterial and fungal communities were assessed via stool samples collected at baseline and following 4 weeks of once-a-day BIOHM consumption. Mycobiome analysis following probiotic consumption revealed an increase in Ascomycota levels in enrolled individuals and a reduction in Zygomycota levels (p value < 0.01). No statistically significant difference in Basidiomycota was detected between pre- and post-BIOHM samples and control abundance profiles (p > 0.05). BIOHM consumption led to a significant reduction in the abundance of Candida genus in tested subjects (p value < 0.013), while the abundance of C. albicans also trended lower than before BIOHM use, albeit not reaching statistical significance. A reduction in the abundance of Firmicutes at the phylum level was observed following BIOHM use, which approached levels reported for control individuals reported in the Human Microbiome Project data. The preliminary results from this clinical study suggest that BIOHM is capable of significantly rebalancing the bacteriome and mycobiome in the gut of healthy individuals, suggesting that further trials examining the utility of the BIOHM probiotic in individuals with gastrointestinal symptoms, where dysbiosis is considered a source driving pathogenesis, are warranted.     

Amelioration of Diabetic Nephropathy by Targeting Autophagy via Rapamycin or Fasting: Relation to Cell Apoptosis/Survival

Autophagy has been demonstrated to have a beneficial effect on diabetic nephropathy (DN). Rapamycin, an inhibitor of mTOR, was shown to stimulate β-cell autophagy. However, its effects on preventing or ameliorating DN is unclear, and its effects are worth studying. As fasting is now an attractive protective strategy, we aim to compare its effect to rapamycin effects on pancreatic and renal cells. Twenty-eight adult male Wistar Albino rats were randomly divided into four groups, using streptozotocin (STZ) to induce diabetes mellitus (DM). Autophagy was induced by two ways; rapamycin or fasting. The extent of autophagy and apoptosis were investigated by measuring the level of LC3B and p53 proteins, respectively, in pancreatic and kidney tissues using Western blotting (WB) technique and imaging the renal cells under transmission electron microscope. The efflux transporter P-glycoprotein was quantified by WB as well. Rapamycin-induced autophagy occurred concurrently with apoptosis. On the other hand, fasting supported P-glycoprotein recovery and renal cell survival together with disabling β-cells apoptosis. In conclusion, this study provides a potential link between rapamycin or fasting for the cross-regulation of apoptosis and autophagy in the setting of cell stress as DN. Unlike rapamycin, fasting enhanced the active expression of ABCB1 efflux protein, providing insights on the potential ameliorative effects of fasting in DN that require further elucidation.