bluuebunny/crossref_metadata_2025 huggingface dataset:
Evenly Re‐distributing Daily Dietary Protein Intake Does Not Augment Changes in Body Composition and Cardio‐metabolic Health Indexes
Authors: Hudson Joshua L. , Kim Jung Eun , Paddon‐Jones Douglas , Campbell Wayne W.
Published: November 2023

Limited, inconsistent research suggests that re‐distributing total protein intake from one very high‐protein meal daily, to multiple moderately‐high protein meals, improves 24 h muscle protein synthesis and over‐time, and may promote positive changes in indexes of body composition and cardio‐metabolic health. We sought to assess the effects of within‐day protein intake distribution on dietary energy restriction and resistance training‐induced changes in body composition, including skeletal muscle size, and cardio‐metabolic health indexes. In a randomized, parallel‐design study, 41 men and women (mean ± SEM; age: 35 ± 2 y; BMI: 31.5 ± 0.5 kg/m2) performed resistance training 3 times/wk for 16 wk concurrent with consuming a controlled, energy‐restricted (750 kcal/d below estimated energy requirements) diet. Subjects consumed 90 g protein/day in either a skewed (10 g breakfast, 20 g lunch, 60 g dinner; n=20) or even (30 g each at breakfast, lunch, and dinner; n=21) distribution pattern. Consuming an energy‐restricted diet resulted in reductions of fasting state body mass (LSmean ± SE; −7.9 ± 0.6 kg), lean mass (−1.0 ± 0.2 kg), fat mass (−6.9 ± 0.5 kg), serum glucose (−2.9 ± 1.2 mg/dL), HOMA‐IR (−1.3 ± 0.2), total cholesterol (−21 ± 4 mg/dL), and triglycerides (−26 ± 7 mg/dL). However, right mid‐thigh muscle area (cm2) did not change over time. Collectively, the within‐day distribution of protein intake did not differentially affect body composition, muscle size, or cardio‐metabolic health index responses over time. Consuming an equivalent amount of protein daily distributed evenly (30‐30‐30 g/meal) versus skewed (10‐20‐60 g/meal) during dietary energy‐restriction combined with resistance training does not confer additional benefits to fat loss, lean mass retention or cardio‐metabolic health outcomes in young adults with overweight or obesity.

Support or Funding Information

Pork Checkoff; American Egg Board‐Egg Nutrition Center; National Dairy Council; Beef Checkoff; and the National Institutes of Health, Indiana Clinical and Translational Sciences Institute, Clinical Research Center, Grant # UL1TR001108.
A class of models to estimate energy requirements
Authors: Plucker Andrew , Powell Michael , Broskey Nick , Martin Corby K , Heymsfield Steven B , Schoeller Dale , Thomas Diana , Redman Leanne
Published: November 2023

Background

Accurately estimating energy requirements represents a standard activity for developing effective diet and exercise interventions. Mathematical models that predict energy requirements as a product of physical activity level (PAL) and a resting energy expenditure (REE) formula is a commonly applied method to provide a first pass estimate. These estimates require knowledge of an individual's PAL and an accurate prediction of REE. Access to different anthropometric data or body composition and even REE measurements can improve and personalize predictions without making assumptions involving PAL.

Methods

Total energy expenditure measured by DLW and metabolic chamber from 733 subjects obtained from compiled study database of baseline measurements measured at Pennington Biomedical Research Center was applied as two different output variables. The DLW measures were applied to develop free‐living energy requirement models and the chamber data was applied to develop in‐residence energy requirement models. Twenty‐eight different linear regression models were developed that included different combinations of input variables that may be accessible to investigators and clinicians. The input variables were age, height, gender, weight, waist circumference, fat mass, fat free mass, and REE. The simplest model predicting DLW measured energy expenditures was validated on the Institute of Medicine DLW database (N=473) and compared to the product of 1.6 and the Mifflin St. Jeor prediction of REE.

Results

The adjusted R2 values for the models predicting free‐living energy requirements in males ranged from 0.65 with minimal covariates of age, height, and weight to 0.73 in models that included body composition or REE. For females adjusted R2 ranged from 0.68 to 0.74. The adjusted R2 values for the models predicting in‐residence energy requirements were lower (males 0.43–0.45, females (0.32–0.33). The bias in the newly developed models was −95±461 kcal/d while the bias obtained from using 1.6 times REE predicted by Mifflin St. Jeor yielded a bias of −315±444 kcal/d.

Conclusions

The newly developed class of models offers an improved alternative to estimating a PAL value and energy requirements using REE formulas. Additionally, when available, the models include additional covariates that improve predictions even further.
A comparison of the effect of protein vs. carbohydrate consumption on human energy balance in free‐living conditions
Authors: Gibson Madeline Jeansonne , Chatindiara Idah , Ironuma Bridget J , Dawson John A , Ovalle Fernando , Allison David B. , Dhurandhar Emily J
Published: November 2023

Background

Several clinical studies have demonstrated beneficial effects of protein on satiety and reduced energy intake acutely, whereas some evidence suggests carbohydrate consumption may lead to positive energy balance. However, it is unclear if these findings have high external validity, or applicability and effectiveness in free‐living settings. Our objective was therefore to compare the effect of consumption of egg protein to consumption of carbohydrate on energy balance in an ad‐libitum, free‐living setting.

Methods

A double blinded, randomized crossover study with a two‐week washout period was conducted. The treatments were mandatory consumption of protein supplied as an egg protein shake (PS) or mandatory consumption of a maltodextrin shake (CS)(23, 32, or 42 g dose with each meal, individualized depending on basal metabolic rate) each for 5 days. The shakes were consumed before breakfast, lunch and dinner. All meals were consumed in a laboratory setting in a buffet format, and food intake was measured covertly by weighing before and after consumption. Physical activity energy expenditure was measured using accelerometers for the entire duration of the treatment period, and satiety and hunger were measured using self‐reported visual analogue scales (VASs). Missing values were addressed via multiple imputation based on available intake data within and across subjects. Changes in energy balance, calculated based on energy intake and total energy expenditure (from resting metabolic rate and accelerometer‐measured physical activity), were compared across PS and CS weeks using a linear mixed effects model, accounting for the crossover design. VAS measures were compared via comparisons of area under the curve (AUC).

Results

There was no evidence for significant carryover effects in any analysis. Change in energy balance on CS was higher than PS (+115 kcal/week) but this amount is not statistically significant; the corresponding p‐value was always at least p = 0.31 in each of our 1000 imputations. Similarly, subjects reported weekly changes of more hunger (+723 mm2), less fullness (−549 mm2), more desire to eat (+634 mm2) and ability to eat much more (+713 mm2) while on PS when compared to CS, but these differences were not statistically different from 0 (all p > 0.20).

Conclusion

The effects of mandatory consumption of egg protein on energy balance in a free‐living setting do not differ from the effects of mandatory consumption of maltodextrin over a 5‐day period. This study does not support that eating high‐protein foods in this context would have a beneficial effect on energy balance, when compared to consuming high carbohydrate foods.

Support or Funding Information

This work was funded by the Egg Nutrition Center.
Tackling Gaps in The Implementation of The Lebanon National Salt Iodization Policy
Authors: Akik Chaza , El‐Mallah Carla , El‐Jardali Fadi , Zimmermann Michael , Ghattas Hala , Obeid Omar
Published: November 2023

Despite a universal salt iodization law in Lebanon, a recent nationally representative study highlighted that the median urinary iodine concentration in schoolchildren was only 66 μg/L and almost 75% of 6 to 10 year old schoolchildren have mild iodine deficiency. As a result, identifying and addressing the existing gaps in the implementation of the salt iodization process became a crucial necessity. Herewith, we document the knowledge translation process initiated by researchers together with an international non‐governmental organization, and its subsequent impact.

In collaboration with the Knowledge to Policy (K2P) Center, a policy brief was developed to synthesize the local and international evidence and viable policy options. Despite a mandatory salt iodization law issued by the Lebanese government, implementation barriers including loopholes in the law, outdated infrastructure and capacity of salt producers were identified. The law was also poorly evaluated and monitored by related authorities. Three policy options to address the issue were put forward 1) amending the law; 2) strengthening implementation of salt iodization by ensuring adequate standards, infrastructure and capacity; and 3) monitoring and evaluating the implementation of the law at the production, retail and consumer levels by building an efficient system for routine data collection.

The policy brief was shared with relevant stakeholders and a national policy dialogue was held. Based on the policy deliberations, the following recommendations were put forward and implemented: (1) the Ministry of Public Health issued a ministerial decree providing clarifications on the existing law, including standards and mechanisms for monitoring and evaluation; (2) with support from international actors, salt producers were trained on monitoring protocols; (3) a one‐year supply of potassium iodate for fortification has been guaranteed by the United Nations Children's Fund.

Once all actions are in place, program evaluation will be done at one year post‐implementation through a market analysis of salt samples and the reassessment of national level iodine status. However, further engagement from governmental authorities is needed to develop mechanisms to ensure sustainability of external monitoring and overcome structural barriers salt producers encounter in importing potassium iodate.

Support or Funding Information

This work was funded by the Iodine Global Network and supported by the K2P Center.
The Efficacy of an Urban Farm Intervention Based on the Stages of Change for a Low Income Population with Multiple Risk Factors for Diabetes
Authors: Clare Rachel A. , Jenkins James , Humphries Debbie
Published: November 2023

Background

Obesity and chronic disease are highly prevalent in the United States, and adherence to dietary practices that decrease risk is low. Innovative approaches to addressing social, behavioral, and nutritional risk factors are crucially needed. The New Haven Farms' Farm‐Based Wellness program (FBWP) is an innovative approach, which provides regular exposure to and participation in urban agriculture with an aim of increasing fruit and vegetable consumption and decreasing diabetes risk factors.

Objective

To evaluate the FBWP's effect on participants' perceived and actual stages of change, fruit and vegetable consumption, and the relationship between consumption and the stages of change before and after program completion.

Methods

Program participants are recruited from neighborhood community health centers in New Haven, CT and must have at least two diabetes risk factors and income within 200% of the 2010 U.S. poverty line. Participants attend a sixteen‐week on‐farm program integrating farm work, cooking and nutrition education, and receipt of a weekly share of vegetables and some fruits. Participants complete a baseline, weekly and closing assessment that includes anthropometry, blood pressure, household and personal characteristics, the stages of change questionnaire (Lechner, et al.) and a national validated dietary screener to estimate fruit and vegetable intake (NHANES Dietary Screener).

Results

73 participants completed the baseline assessment in 2016, 90 attended at least one weekly session, and 30 completed both the baseline and the final assessment, as well as attending some weekly sessions. At baseline, 22.0% were in precontemplation and contemplation, 70.4% were ready for change, and 7.4% already consumed a mean of 2.6 servings of vegetables per day. For fruit, 25.9% were in precontemplation and contemplation, 51.9% were ready for change, and 18.5% already consumed a mean of 2.8 servings of fruit per day. The mean daily combined fruit and vegetable intake was 3.1 servings both for those who only completed the baseline survey as well as for the 30 participants with baseline and final data. Among the 30 participants with complete data, after program completion daily vegetable servings increased to an average of 2.1 daily servings (p=0.008) and daily fruit servings decreased to 1.5 daily servings. Initial stage of change was associated with an increase in vegetable consumption (p= 0.087), but not in fruit consumption (p=0.658). Individuals in the action/maintenance stage increased vegetable intake an average of 1.4 servings per day, in preparation increased an average of 0.6 servings per day, and in precontemplation decreased an average −0.1 servings per day.

Conclusions

These findings suggest that targeting of the program to individuals in a specific stage of change might increase the overall impact of the program. The FBWP may benefit from integration of behavior change models in programming.
Modulation of Urinary Siderophores by the Diet, Gut Microbiota and Inflammation in Mice
Authors: Xiao Xia , Yeoh Beng San , Saha Piu , Tian Yuan , Singh Vishal , Patterson Andrew D. , Vijay‐Kumar Matam
Published: November 2023

Mammalian siderophores are believed to play a critical role in maintaining iron homeostasis. However, the properties and functions of mammalian siderophores have not been fully clarified. In this study, we have employed Chrome Azurol S (CAS) assay which is a well‐established method for bacterial siderophores study, to detect and quantify mammalian siderophores in urine samples. Our study demonstrates that siderophores in urine can be altered by diet, gut microbiota, and inflammation. C57BL/6 mice, fed on plant‐based chow diets which contain numerous phytochemicals, have more siderophores in the urine compared to those fed on purified diets. Urinary siderophores were upregulated in iron overload conditions, but not altered by other tested nutrients status. Further, germ‐free mice displayed 50% reduced urinary siderophores, in comparison to conventional mice, indicating microbiota biotransformation is critical in generating or stimulating host metabolism to create more siderophores. Altered urinary siderophores levels during inflammation suggest that host health conditions influence systemic siderophores level. This is the first report to measure urinary siderophores as a whole, describing how siderophores levels are modulated under different physiological conditions. We believe that our study opens up a new field in mammalian siderophores research and the technique we used in a novel manner has the potential to be applied to clinical purpose.

Support or Funding Information

This work was supported by grants from the National Institutes of Health (NIH) R01 [Grant number DK097865].
Piglets Are Vulnerable to Intestinal Inflammation and Infection Because of Expression of Low‐Affinity Jejunal Alkaline Phosphatase Activities
Authors: Burello Nicole A. , Archbold Tania , Wang Weijun , Shoveller Anna‐Kate , Fan Ming Z
Published: November 2023

Intestinal alkaline phosphatase (IAP) plays a pivotal role in protecting gut health through catalyzing the hydrolytic dephosphorylation of the lipid A moiety of endotoxin lipopolysaccharides (LPS) and other emblematic members of pathogen‐associated‐molecular patterns (PAMPs) such as ATP, ultimately detoxifying endotoxins and preventing gut dysbiosis and disorders. Characterization of IAP hydrolytic activities, including enzyme affinity (Km) and the maximum enzyme activity (Vmax), has been reported in pigs and other mammalian species by using the chromogenic synthetic substrate P‐nitrophenyl phosphate (pNPP) rather than using relevant physiological substrates such as ATP and LPS under the physiological pH conditions. This study was conducted to determine kinetics of young porcine jejunal alkaline phosphatase in hydrolyzing the dephosphorylation of ATP, the lipid A moiety of LPS and pNPP at pH 7.4 and 37 °C. Jejunal samples were collected from four 10‐day old Yorkshire pigs and were pulverized to be homogenous under liquid N2. The LPS kinetic experiments were conducted with 11 gradient concentrations of LPS, isolated from E. coli 0111:B4, ranging 0 – 5 mg/mL in incubation media. The ATP kinetic experiments were conducted with 11 gradient concentrations of ATP, ranging 0 – 5 mM in incubation media. Free phosphorus (Pi) release was measured by using the PiColorLock detection reagent (Innova Biosciences). Contributions of the non‐specific background Pi associated with ATP and LPS in the time course and the kinetic experiments were corrected. The pNPP kinetic experiments were conducted with 16 gradient concentrations of pNPP, ranging 0 – 6 mM in incubation media. All enzymatic incubations were conducted with 19 – 27 μg of the homogenized porcine jejunal protein. The kinetics (parameter estimates ± SE, P < 0.05) of young porcine jejunal alkaline phosphatase activities were obtained, including Vmax values of 2.5 ± 0.4 ug/mg protein·min (n = 11), 1.1 ± 0.2 ug/mg protein·min (n = 11), and 0.12 ± 0. 01 umol/mg protein·min (n = 16); and Km values of 1.255 ± 0.498 mM (n = 11), 2.388 ± 0.794 mg/mL (n = 11), and 0.145 ± 0.036 mM (n = 16), for the hydrolysis of ATP, LPS and pNPP, respectively. Our results reveal the young porcine IAP Km value 41 times higher in comparison with reported Km value of human IAP in dephosphorylating ATP, 3 times higher when compared with reported human IAP dephosphorylating LPS, and 5 times higher when compared with reported human IAP hydrolyzing pNPP. Results of this study show that young porcine jejunal IAP activity affinity is several fold lower in detoxifying endotoxin LPS the other emblematic PAMPs member ATP in comparison with humans, suggesting that young pigs are susceptible to gut inflammation and infection.

Support or Funding Information

NSERC Discovery Program of Canada and Metagen Enzyme Corporation
The Effects of Instant Coffee on the Myotatic Reflex During Bench Pressing: A Gender Comparison
Authors: Ruffner Kayla , DiFiore Brittany , Smith Bridget , Trivisonno Abigail , Slack Grant , Cooper Cyrena , Lowery Lonnie
Published: November 2023

Coffee is widely consumed by exercising men and women throughout the world. Previous work from our laboratory suggests coffee enhances the stretch reflex during bench pressing. In partial contrast, Carins and colleagues (2003) were able to enhance the stretch reflex in men but not women during a non‐exercise intervention using glutamate. Whether coffee differently affects reflexes in each gender during explosive exercise remains unclear. The purpose of this study was to compare the neuromuscular reflexes of the upper body between sexes after consuming two test beverages. We hypothesized that men would have a greater enhancement of reflexive bench press performance compared to women after ingesting Via Instant Coffee (VIA) compared to decaffeinated coffee (DCF). In a blinded, crossover experimental design, 34 resistance trained individuals were tested at 50% of their one repetition maximum (1RM) on the Smith Bench Press one hour after ingesting 525 mL VIA or DCF. Force (FOR) and time to peak power (TtPP) were measured using the Smith machine and Ballistic Measurement System (BMS, Innervations Inc, South Australia). After repeated measures ANOVA and Newman‐Keuls post hoc testing, absolute bench press FOR was enhanced by prior stretch in the DCF condition (mean ± SD) in men (806.7 ± 206.3 N vs. 1040.5 ± 287.3 N; p≤0.05) and women (318.6 ± 65.2 N vs. 406.9 ± 113.4 N; p≤0.05) with larger enhancement after consuming VIA (men 806.0 ± 222.9 N vs. 1114.1 ± 343.8 N; p≤0.05 and women (327.0 ± 67.1 N vs. 403.9 ± 89.5 N; p≤0.05). Statistical correction for body mass, however, abolished sex differences (p≥0.05). TtPP enhancement from prior stretch tended to differ in the DCF condition in men (328 ± 70 ms vs. 216 ± 80 ms) and in women (378 ± 90 ms vs. 318 ± 110 ms; interaction p=0.096). This trend reached statistical significance after consuming VIA in men (315 ± 70 ms vs. 194 ± 80 ms; p≤0.05) and in women (367 ± 100 ms vs. 302 ± 100 ms; p≤0.05). We conclude that the stretch reflex enhances explosive bench press performance in both sexes with men gaining greater absolute gains than women, particularly after coffee consumption. Future studies that compare sexes in the bench press are cautioned to adjust force measures for body mass differences.
The CXCR3‐LFA1‐ICAM1 Axis Regulates T Cell Cardiotropism and Maladaptive Cardiac Remodeling in Heart Failure
Authors: Salvador Ane , Nevers Tania , Velazquez Francisco , Aronovitz Mark , Alcaide Pilar
Published: November 2023

Left ventricular (LV) dysfunction and heart failure (HF) are associated in humans and mice with increased circulating chemokines CXCL9 and CXCL10. T cells expressing their receptor, CXCR3, are expanded in the circulation of HF patients as compared to non‐HF, and are associated with disease progression. CXCR3 signaling upon chemokine recognition in T cells results in integrin activation and T cell adhesion to endothelial cells. We recently reported that the integrin ligand ICAM1 is significantly upregulated in the LV endothelium in mice with HF, and that ICAM1−/− mice have decreased LV T cell recruitment and preserved HF. Thus, we hypothesized that chemokine signaling through CXCR3 regulates integrin dependent adhesion to ICAM1 and contributes to T cell recruitment into the LV and HF. We used the mouse model of Thoracic Aortic Constriction (TAC) to induce LV remodeling and HF in WT mice, and in vitro differentiated effector T cells to evaluate their integrin dependent affinity to ICAM1 in the presence of chemokines under shear flow conditions. CXCL9 and CXCL10 were upregulated in WT mice in response to 2 and 4 weeks TAC as compared to Sham, correlating with ICAM1, IL‐6 and IL‐1β upregulation. CXCR3+ lymphocytes were expanded in the mediastinal lymph nodes as early as 1 week post TAC and remained increased 2 and 4 weeks post TAC in WT mice, as compared with Sham. CXCR3+ T cells were recruited to the LV 4 weeks post TAC and expressed higher levels of the integrin LFA1, the main ligand of ICAM1, in contrast to CXCR3‐T cells. Moreover, both CXCL9 and CXCL10 induced adhesion of CXCR3+ T cells to ICAM1 under flow conditions in vitro in an LFA‐1 dependent manner, and CXCL9 effect was greater when compared to CXCL10 induced CXCR3+ T cell adhesion to ICAM1. Our data supports a CXCR3‐LFA1‐ICAM1 axis being involved in T cell mediated pathological cardiac remodeling, representing a novel pathway that could be targeted in non‐ischemic HF.

Support or Funding Information

National Institutes of Health NIH RO1 HL123658 and American Heart Association Grant in Aid‐AHA GIA 13GRNT 14560068
Development of a biodegradable retinal cell graft for the treatment of retinal degenerative blindness
Authors: Gibson‐Corley Katherine N. , Worthington Kristan S. , Thompson Jessica R. , Kaalberg Emily , Jiao Chunhua , Mullins Robert F. , Stone Edwin M. , Sohn Elliott H. , Tucker Budd A.
Published: November 2023

Background

Vision loss in inherited retinal diseases such as age‐related macular degeneration or retinitis pigmentosa result from death of the photoreceptor cells of the outer retina. Subretinal transplantation of induced pluripotent stem cell‐derived retinal progenitor cells (iPSC‐RPCs) has been found somewhat effective in restoring vision in blind animals, although survival of transplanted cells is poor. Polymeric support scaffolds have been shown to greatly enhance cellular survival and integration post‐transplantation, however attempts to materialize this concept have been largely unsuccessful for two reasons: 1) mechanical mis‐matching between material and the host retina and 2) improper cell packing. The goal of this study was to determine the ideal polymer fabrication parameters that promote optimal cell packing and biocompatibility.

Methods

Methacrylate‐functionalized PCL scaffolds were polymerized with either Irgacure 651 or Irgacure 369 using two‐photon polymerization. Compressive modulus was measured using dynamic mechanical analysis. Four‐month‐old Yucatan mini pigs (n=10, 5 with Irgacure 651 and 5 with Irgacure 369) received 5mm diameter scaffolds containing iPSC‐RPCs via subretinal transplantation. Animals were examined clinically at 1 month post‐surgery and were subsequently sacrificed for histological analysis of the retina.

Results

PCL scaffolds were successfully generated using both photoinitiators. Scaffolds generated using Irgacure 651 cured rapidly and had a tendency to curl. Both Irgacure 651‐ and Irgacure 369‐generated scaffolds supported attachment and survival of human iPSC‐RPCs and both were successfully transplanted into the porcine subretinal space. At one month post‐transplantation, 50% of the retinas that received the Irgacure 651 scaffolds were attached. In the animals that received Irgacure 369 scaffolds all of the retinas reattached with no signs of ocular inflammation. In these animals retinal integrity, including maintenance of photoreceptor outer segments, was maintained.

Conclusion

PCL scaffolds support the maintenance of human iPSC‐RPCs and were biocompatible in the pig retina. These scaffolds may be ideal for retinal transplantation.

Support or Funding Information

Wynn Institute for Vision Research
Electrospun Nanofibers as Scaffolds for Wound Healing
Authors: Nwachukwu Cynthia C , Einstein George P , Tulp Orien L
Published: November 2023

A chronic non‐healing wound presents a challenging and escalating problem which exposes patients to a high risk of infection. Current treatment narrows in on wound dressings that keep an environment of equal moisture and gas balance. Antibacterial dressings with a responsive system to trigger release only when infection is present and methods stimulating cell development to promote healing have now been developed. However few of these dressings are effective in simultaneously treating infection and promoting wound healing. Electrospinning is a unique simple, cost‐effective, and reproducible approach using electrostatic forces that produce fine fibers from both synthetic and natural polymer solutions to address these specific wound challenges. Electrospun fibers provide high‐surface area, micro‐porosity, and the ability to load drugs or other biomolecules into the fibers. These fibers are being studied and have been developed because they hold considerable promise for realizing some advantages of nanostructured materials. Electrospun materials have been used as scaffolds for tissue engineering for a number of years, but there is little literature on the interactions of fibers with co‐cultures of cells and bacteria. This study presents an introduction to polymer nanofiber electrospinning, focusing on the use of natural proteins and synthetic peptides. We summarize key physical properties of protein‐based and peptide‐based nanofiber mats, survey biomedical applications of these materials, identify key challenges, and outline future prospects for development of the technology for tissue engineering and drug delivery to address the development of smart multifunctional wound dressings capable of treating infection and healing chronic wounds simultaneously.

Support or Funding Information

Supported by Institutional Resources of USAT, Einstein Medical Institute, and Biomaterials Laboratory, University of South Florida
Distinguishing the Central Pathways of the Inflammatory Reflex from those of Fever
Authors: Martelli Davide , Farmer David George , Yao Song Tieng , McKinley Michael Joseph , McAllen Robin Michael
Published: November 2023

In response to a systemic inflammatory challenge, the CNS orchestrates several stereotyped responses. First, the hypothalamic‐pituitary‐adrenal axis is activated. Second, there is fever. Third, the splanchnic anti‐inflammatory pathway (SAIP), the efferent arm of the inflammatory reflex, is activated to inhibit an excessive release of pro‐inflammatory markers (Martelli et al. Exp Physiol 2016;101(10):1245–1252). The first two responses, dependent on prostaglandin synthesis, are blocked by cyclooxygenase (COX) inhibitors (Garcia‐Bueno et al. J Neurosci 2009;29(41):12970–81; Steiner et al. Am J Phys Regul Integr Comp Physiol 2009;297(2):R485–94). We report here that the SAIP is not. In rats, bilateral section of the splanchnic nerves enhanced the plasma levels of TNF‐α 90 mins after i.v. lipopolysaccharide (LPS; 60 μg/kg) several‐fold, and this remained the case in animals treated with the COX inhibitor diclofenac (5mg/kg × 2 injections i.v., before and 45 minutes after LPS injection), even though fever was blocked. Activation of the SAIP by LPS was evidently unaffected by COX inhibition sufficient to block fever. Using this knowledge, we set out to identify which sympathetic premotor neurons drive the SAIP, removing the confound of fever. In preparatory surgery, the retrograde tracer Fast Blue was injected around the IML of the lower thoracic cord in 18 rats. Two weeks later, the femoral vein was cannulated and the animal allowed to recover for 48 hours. Diclofenac (5mg/kg) was given i.v. 10 mins before LPS (60 μg/kg, i.v.). 150 mins later rats were deeply anesthetized and perfusion‐fixed. Brains were frozen sectioned and processed immunohistochemically for Fos. Double fluorescence microscopy revealed fast blue labeled (spinally –projecting) and Fos‐labeled neurons. The only site found to show double labeled neurons was a sub‐area of the hypothalamic paraventricular nucleus. These may be the premotor neurons that drive the SAIP.

Support or Funding Information

DM is supported by NHMRC (GNT1098887) and an IBRO award. STY is supported by grants from the NHMRC (GNT1079680). The Florey Institute of Neuroscience and Mental Health acknowledges the strong support from the Victorian Government and in particular the funding from the Operational Infrastructure Support Grant
The impact of neuroanatomy on my career: from adult neurogenesis to in vitro disease modelling
Authors: Merkle Florian
Published: November 2023

I am truly honored to be selected for this year's Young Investigator Award. It only seems appropriate to highlight how insights from neuroanatomy have shaped my career. I have always marvelled at how something as fantastically complex and beautiful as the brain could develop, so I pursued this curiosity by studying adult neurogenesis as a PhD student. By observing and taking advantage of the unique anatomical characteristics of early stem cells, my colleagues in Arturo Alvarez‐Buylla's laboratory and I demonstrated the lineage relationship of embryonic and adult neural stem cells. We further labelled stem cells in different regions of the adult neurogenic niche to show that they are better classified as distinct and restricted progenitors than homogeneous multipotent stem cells, and in the process identified four novel types of adult‐born interneurons. As a postdoctoral fellow, I used insights from developmental biology and anatomy to develop a method to differentiate human pluripotent stem cells into specific populations of hypothalamic neurons that regulate essential behavioral and physiological functions, such as feeding and sleep. In my own laboratory, we are now using single cell RNA sequencing and cellular assays to explore the functional heterogeneity of in vitro‐derived neurons. We then use these insights to map neuronal sub‐populations back to the mouse and human brain, where their anatomical location provides essential clues as to their likely function in the organism. Although new tools and evolving research interests have altered the trajectory of my career, neuroanatomy has provided a constant link.

Support or Funding Information

The research presented in this talk was supported by the National Science Foundation, the National Institutes of Health, the Jane Coffin Childs Memorial Fund for Cancer Research, the Harvard Stem Cell Institute, and the Howard Hughes Medical Institute. Current funding sources include the Wellcome Trust, the Medical Research Council, the Academy of Medical Sciences, and the University of Cambridge.
Promising effects of laser therapy and the natural latex protein on sciatic nerve crushed. Ultrastructural, sensory and functional study
Authors: Dias Fernando Jose , Simões Ana Leda Bertoncini , de Cássia Carmo Elisabete , Cury Diego Pulzatto , Almeida Sonia Regina Yokomizo , Dias Paula Elisa , Fazan Valeria Paula Sassoli
Published: November 2023

Treatment of lesions that affect peripheral nerves still presents a challenge, in this context the low‐level laser (LLL) and the protein of the natural latex (Hevea brasiliensis, F1) showed positive effects.

The aim of this study was to analyze the effects of LLL associated with F1 in crush injuries of the sciatic nerve (15 kgf, 10') in rats. Six groups (n=24) of rats (♂, 250 g): C ‐ Control; E ‐ Exposed nerve without crushing; Le ‐ injured nerve without treatment; L ‐ injured nerve LLL irradiation (15 J/cm2, 780 nm); F1 ‐ injured nerve with application of F1 protein (0.1%); and LF1 ‐ injured nerve with the F1 protein and LLL irradiation. After 1, 7, 14, 28, 56 and 180 days of nerve injury of sensory and functional analyzes were performed: 1. mechanical allodynia (von Frey); 2. Pressure testing the paw (Forceps); 3. Grip strength test and 4. Motor function (SFI). After 3, 15, 57 and 181 days of nerve injury, the animals of each subgroup (n=6) submitted to intracardiac perfusion (2.5% glutaraldehyde in cacodylate buffer 0.1 M, pH 7.2), the sciatic nerve samples were collected and processed to ultrastructural analysis.

The results revealed that the controlled nerve compression (15 kgf) was intense, thus spontaneous recovery was not enough to restore the normal characteristics of the sciatic nerve allowing to evaluate the effects of proposed therapies. The three treatment protocols proposed LLL, F1 and LLL+F1 revealed improvements in ultrastructural characteristics when compared with the untreated injured animals. It was noted correlation between these data and those functional sensory and motor, which also showed improvements with the treatments applied. All treatments applied resulted in improvements in the characteristics and tests evaluated, the time in all cases was an important factor in this recovery however in some functional tests there was an acceleration of improvements, and the animals that received F1 associated or not to LLL had generally the best results.

Support or Funding Information

Funding: FAPESP 2014/07253‐4
Unraveling gene regulatory mechanisms in meiotic differentiation
Authors: Brar Gloria , Cheng Ze , Van Dalfsen Kelsey , Eisenberg Amy , Hollerer Ina
Published: November 2023

Meiosis is a well‐conserved program of cellular differentiation. We performed genome‐wide measurements of translation using ribosome profiling through meiosis in budding yeast to better understand the molecular basis for the full cellular restructuring that accompanies meiotic chromosome segregation. This first global glimpse at translation through a developmental process revealed great complexity to the protein complement of these differentiating cells, both in the number and the structure of expressed genes. Nearly every gene in the yeast genome was translated in meiotic cells in a strongly stage‐specific manner, including thousands of genes with no previously known meiotic role. Prominent among these was key regulators of conserved stress response pathways, suggesting that meiotic cells coopt such pathways for programmed cellular remodeling. Translational regulation contributed broadly to the timing and levels of protein synthesis through several disparate and complex mechanisms, including use of upstream Open Reading Frames Additionally, meiotic translation of thousands of novel short ORFs was observed, including on transcripts previously thought to be noncoding and as alternate translation products from characterized transcripts, expanding our view of what constitutes a coding region even in the most well annotated eukaryotic genome.

Support or Funding Information

I am grateful for the NIH funding (DP2‐GM119138 and P50‐GM102706) that currently supports research in my lab, along with investigator awards from the March of Dimes, Alfred P. Sloan Foundation, and Pew Charitable Trusts.
Ultrafast recycling of synaptic vesicles
Authors: Watanabe Shigeki
Published: November 2023

Neurons can fire at extremely high rates. To sustain neurotransmission, synaptic vesicles must be recycled locally at synapses. Two models for synaptic vesicle endocytosis have been put forward based on the morphological studies in frog neuromuscular junctions. Heuser and Reese proposed that endocytosis occurs via a slow mechanism using clathrin scaffolds. Ceccarelli and his coworkers proposed a fast mechanism, kiss‐and‐run. Since then, many studies have sought to identify the mechanism for synaptic vesicle endocytosis. However, instead of resolving the issue, conflicting evidence has accumulated over the years. The molecular studies have suggested clathrin and clathrin‐associated proteins are essential. However, the kinetics studies have suggested that both forms co‐exist. Our data identify a third pathway that is fast, but requires clathrin to regenerate vesicles.

To investigate how endocytosis takes place, we developed a method, ‘flash‐and‐freeze’ that couples optogenetic stimulation with rapid high‐pressure freezing and captures endocytosis at millisecond temporal resolution. To our surprise, vesicle membrane is recovered via ultrafast endocytosis within 100 ms following a single stimulus. The large endocytic vesicles then fuse to form an endosome and are resolved by clathrin into synaptic vesicles in 5–6 s. When experiments are performed at 20°C instead of 37°C, ultrafast endocytosis fails, and clathrin regenerates synaptic vesicles directly from plasma membrane. These results suggest that recycling of synaptic vesicles is normally a rapid two‐step process: ultrafast endocytosis that removes excess membrane from the surface and then clathrin‐mediated biogenesis of synaptic vesicles from endosomes.

Support or Funding Information

SW is supported by JHU startup fund and JHU discovery fund.
Malaria derived extracellular vesicles influence human neutrophils function
Authors: Babatunde Kehinde Adebayo , Walch Michael , Fellay Isabelle , Kharoubi‐Hess Solange , Filgueira Luis , Ghiran Ionita , Mantel Pierre‐Yves
Published: November 2023

Introduction

A dysfunctional innate immune response is believed to provide immune evasion of the malaria parasites, but also to cause increased susceptibility to bacterial infections. Neutrophils are the most abundant cells found in the blood circulation in direct contact with parasite infected red blood cells (iRBCs). However neutrophils population with reduced oxidative burst activities are present during malaria infection. These observations suggest that neutrophil responses are fundamentally defective in malaria patients. Extracellular vesicles (EVs) are iRBCs derived vesicles and contain both parasite and host materials, including microRNAs. In this present work we investigated, how EVs modulate neutrophil response.

Results

Interestingly, we have reported that malaria EVs contain miR451a, a miRNA that is known to regulate neutrophil activity. We have also monitored the uptake of EVs by neutrophils by fluorescence microscopy and real‐time polymerase chain reaction techniques. To address the role of miR451, we investigated the influence of malaria‐induced EVs on human neutrophil functions in vitro and the effect of miR451a on the transcriptional response in differentiated HL‐60 cells to bacterial infections. We demonstrated that EVs inhibit neutrophil function by inhibiting neutrophils ability to produce ROS and suppression of cytokine secretion. The neutrophils were also affected in their bactericidal activity.

Conclusion

Our data indicate that malaria EVs deliver miR‐451 into neutrophils to interfere with their capacity to kill bacteria. We describe a new mechanism of cellular communication between parasites and the host immune system. While EVs might increase tolerance to the parasites, they dramatically affect the resistance to a co‐infection by bacteria. The elucidation of the immune regulatory role of EVs might lead to the development of new diagnostic tools and therapies.
Phosphoinositide conversion in the endolysosomal system
Authors: Haucke Volker , Ketel Katharina , Wallroth Alexander , Marat Andrea L , Lo Wen‐Ting , Schultz Carsten
Published: November 2023

Phosphoinositides (PIs) form a minor class of phospholipids with crucial functions in cell physiology, ranging from cell signalling and motility to a role as signposts of compartmental membrane identity. Phosphatidylinositol 3‐phosphates are present at the plasma membrane and within the endolysosomal system, where they serve as key regulators of both cell signalling and of intracellular membrane traffic. In my talk I will cover our recent advances in the analysis of the metabolic pathways that regulate cellular synthesis of PI 3‐phosphates at distinct intracellular sites and discuss the mechanisms by which these lipids regulate cell signalling and membrane traffic. Finally, I will provide a framework for how PI 3‐phosphate metabolism is integrated into the cellular network.

Support or Funding Information

Deutsche Forschungsgemeinschaft (DFG), TRR186, SFB740
Nuclear envelope rupture is induced by actin‐based nucleus confinement
Authors: Hetzer Martin , Hatch Emily
Published: November 2023

Repeated rounds of nuclear envelope (NE) rupture and repair have been observed in laminopathy and cancer cells and result in intermittent loss of nucleus compartmentalization. Currently, the causes of NE rupture are unclear. We show that NE rupture in cancer cells relies on the assembly of contractile actin bundles that interact with the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex. We found that the loss of actin bundles or the LINC complex did not rescue nuclear lamina defects, a previously identified determinant of nuclear membrane stability, but did decrease the number and size of chromatin hernias. Finally, NE rupture inhibition could be rescued in cells treated with actin‐depolymerizing drugs by mechanically constraining nucleus height. These data suggest a model of NE rupture where weak membrane areas, caused by defects in lamina organization, rupture because of an increase in intranuclear pressure from actin‐based nucleus confinement.
Mechanisms of Zinc Metallostasis in Bacterial Pathogens
Authors: Giedroc David , Capdevila Daiana A. , Martin Julia E. , Edmonds Katherine A. , Wu Hongwei
Published: November 2023

First‐row late d‐block transition metals from Mn to Zn play critical and distinct roles in many aspects of cellular metabolism. In bacterial pathogens, metalloregulation of transcription underscores physiological adaptation to host‐mediated transition metal starvation and toxicity, required to maintain metal homeostasis. In zinc (Zn) homeostasis, a pair of metal‐sensing transcriptional repressors regulate the transcription of metal uptake and efflux transporters, where Zn typically allosterically activates or inhibits, respectively, DNA operator‐promoter binding. We have interests in the molecular underpinnings of allostery in metalloregulatory proteins, transition metal transporter function and the impact that dysregulation of the process has on cellular physiology. In the model Gram‐positive respiratory pathogen, Streptococcus pneumoniae, the Zn uptake regulator is the multiple antibiotic resistance repressor (MarR)‐family protein, adhesin‐competence repressor (AdcR), while the efflux regulator is a novel tetracycline repressor (TetR) family protein, streptococcal zinc activator (SczA). These are interesting regulatory targets with which to probe allosteric regulation of DNA binding by metals since in the vast majority of MarR and TetR repressors, the regulatory ligand functions as an allosteric inhibitor of DNA binding (+ΔGc); in both AdcR and SczA, the sign on the allosteric coupling free energy, ΔGc, is negative or favorable. NMR solution structural studies of both backbone and methyl side‐chain flexibility as a function of allosteric state in AdcR will be discussed, as will NMR studies of AdcR‐regulated Zn uptake transporter, and the Zn efflux regulator SczA. In addition, recent findings of effect of host‐imposed Zn toxicity on pneumococcal physiology will also be presented.

Support or Funding Information

Supported by the US National Institutes of Health grant GM118157.
Substrate Conformational Dynamics in Proteolysis
Authors: Radisky Evette S.
Published: November 2023

Enzyme catalytic power and specificity derive from dynamic interactions between enzymes and their substrates. While considerable effort has been devoted to understanding how enzyme conformational dynamics can affect catalysis, the importance of conformational dynamics within protein substrates has been overlooked. Proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalysis and specificity. Recent work from our research group, focusing on trypsins and chymotrypsins as model proteolytic enzymes, reveals that differing conformational dynamics in otherwise similar protein substrates can profoundly impact proteolysis. In this talk, I will present some of our recent findings, integrating evidence from crystal structures, molecular dynamics simulations, and enzyme kinetics studies, which implicate flexibility and conformational motions of protease substrates as critical determinants of proteolysis. Our studies suggest that specific substrate conformational dynamics have evolved to regulate biological proteolytic events. More generally, our work suggests that we must look beyond enzymes, and examine substrates also, to fully appreciate the importance of protein dynamics in enzyme catalysis.

Support or Funding Information

Supported by NIH grant R01CA154387.
Mechanism and Regulation of RNA Polymerase II
Authors: Kaplan Craig D.
Published: November 2023

Regulated gene expression by RNA Polymerase II (Pol II) is essential for organismal function and underpins development and response to environment in eukaryotes. Alteration to Pol II catalytic activity causes all aspects of transcription from initiation to elongation, termination, and RNA processing to be altered. We have undertaken high‐throughput genetic analyses to dissect the Saccharomyces cerevisiae Pol II active site and determine how individual residues of a critical Pol II domain – the trigger loop (TL) – function. These studies allow us to test models for Pol II function and illuminate the contributions of residues to basic Pol II functions. We have also characterized the mechanisms by which the small molecule thiolutin, a long known transcription inhibitor lacking defined mechanism, alters transcription and yeast cellular physiology. We show that while thiolutin has many in vivo effects, it directly inhibits Pol II activity in multiple ways, and thiolutin‐inhibited Pol II has unique properties not observed for another Pol II inhibitor, alpha‐amanitin. We will discuss how these and other studies allow us to learn about fundamental transcription mechanisms.

Support or Funding Information

NIH GM097260, GM120450

Welch Foundation A‐1763