bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of db‐cAMP on Renal Interstitial Hydrostatic Pressure (RIHP) Response to Renal Medullary Direct Interstitial Volume Expansion (DIVE) in rats
Authors: Flores‐Sandoval Omar , López‐Rodríguez Juan Francisco , Sánchez‐Briones María Eugenia , Calvo‐Turrubiartes Miriam Zarahí , Rodríguez‐Martínez Manuel
Published: November 2023

Renal medullary (rm) DIVE (100 μl infusion in 30 s) with either 0.9% saline solution (SS) or 2% albumin in SS rises slow, progressive and indistinctly the RIHP (FASEB J 27:1110.2,2013). Given the sui generis conformation and architecture of renal medullary interstitium, we assessed if dibutyryl‐cAMP (db‐cAMP, a collagen gel contraction inhibitor) modifies the time curse of RIHP response to rm DIVE with SS. Subcapsular RIHP was continuously measured in anesthetized rats with right nephrectomy, acute left renal denervation, hormonal clamp and control of renal perfusion pressure (100 mmHg) before (Pre: 30 min), during (30 s) and after (Post: 60 min) sham rm DIVE (time CTR group n = 9) and real rm DIVE with either SS (SS group, n = 8) or SS + 5mM db‐cAMP (db‐cAMP group, n = 8). There were no differences among groups in body weight, hematocrit nor plasma protein concentration at any time. The table below shows the absolute values of RIHP (mean ± SEM) before rm DIVE, in the last 10 s of rm DIVE and 20, 40 and 60 min after rm DIVE. 1WANOVA + Tukey test: ¨ P<0.05 vs. CTR group at any time point. 1WRMANOVA: · P<0.008 vs. Before. ANCOVA: there were effect of both RIHP before rm DIVE (as covariate) at any time point (P<0.001) and of group (P<0.02 in the last 10 s of rm DIVE and P<0.0001 60 min after rm DIVE). MANOVA of overall ΔRIHP (after RIHP − before RIHP) data showed time (P<0.0001), group (P<0.0023) and time * group interaction effects (P<0.0013). Continuous measurements of renal medullary blood flow (RMBF, L‐D) in two groups (SS and db‐cAMP) of an independent series showed that for the 60 min after rm DIVE the %ΔRMBF had fallen 34 ± 9 % in SS group while this variable did not change in db‐cAMP group (10 ± 9 %).

CONCLUSIONS

The results suggest that db‐cAMP abolished the RIHP response to rm DIVE with SS by maintaining the baseline RMBF and/or by inhibiting the renal interstitial reactive response to rm DIVE with SS.

Support or Funding Information

C13‐FAI‐03‐36.36/UASLP

Subcapsular RIHP (mmHg)

Group
Before
last 10 s
20'After
40'After
60'After

CTR
1.8 ± 0.1
1.4 ± 0.1
1.8 ± 0.3
1.9 ± 0.3
2.1 ± 0.4

SS
1.5 ± 0.2
2.4 ± 0.6
3.7 ± 0.9•
5.3 ± 1.1◆•
5.7 ± 1.2◆•

db‐cAMP
2.2 ± 0.4
2.5 ± 0.6
2.7 ± 0.5
3.0 ± 0.6♦
2.8 ± 0.5
Effect of Hypoxia on Fibroblast Proliferation in Idiopathic Pulmonary Fibrosis
Authors: Senavirathna Lakmini Kumari , Huang Chaoqun , Liu Lin
Published: November 2023

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and usually lethal lung disease, categorized under idiopathic interstitial pneumonias. The survival rate of this disease is very low because of poor prognosis and limited effective therapies. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix proteins creates a hypoxic condition within the lungs, causing asphyxiation. Therefore, hypoxia is one of the prominent features of IPF. However, the effects of hypoxia on pulmonary fibroblasts are less studied. In this study, we evaluated the effects of hypoxia on pulmonary fibroblast proliferation and associated molecular mechanisms in vitro. Human pulmonary fibroblasts were exposed to normoxia (21% O2) and hypoxia (1% and 5% O2) for 3 and 6 days. Intracellular calcium channels, HIF 1α and HIF 2α were inhibited by specific inhibitors. Cell viability and proliferation were determined. The mRNA expression of Nuclear Factor T cell Activator (NFAT)c1, c2, c3, c4 and 5 were measured by real‐time PCR. Hypoxic conditions did not affect the cell viability. Fibroblast proliferation increased significantly at day 3 and day 6 under both hypoxic conditions compared to normoxic conditions. Calcium channel blocker and HIF 2α inhibitor inhibited hypoxia‐mediated fibroblast proliferation. Hypoxic conditions increased the mRNA expression of NFATc2 and NFATc4. In conclusion, hypoxia induces fibroblast proliferation through calcium channel, HIF 2α and NFATc2/4.

Support or Funding Information

NIH R01 HL116876 and P20GM103648
Vanadium complexes inhibit growth of HT‐29 cells via ROS generation
Authors: Moretti Brandon , McCaffrey Vanessa , Rabquer Bradley James
Published: November 2023

Purpose

Cancer is characterized by uncontrolled cell proliferation caused by a loss of control of cell cycle growth and division. Many chemotherapeutics have been developed to induce apoptosis or inhibit cancer cell growth. Previous studies have evidenced the efficacy of organometallic compounds, notably cisplatin and its derivatives, in inhibiting tumor growth. Further studies have been conducted with other metal elements to assess their potential as anti‐cancer drugs. One in particular is vanadium, which has been shown to elicit effects via multiple biochemical pathways. We previously synthesized novel vanadium complexes with variable functional groups and found that they were able to inhibit growth in breast (MCF‐7) and colon (HT‐29) cancer cell lines to varying degrees. Here, we hypothesized that these complexes were inducing apoptosis via caspase 3/7‐mediated pathways and inducing cell death by an increase in reactive oxygen species generation.

Methods & Results

HT‐29 cells were used as a model of colon cancer, along with a line of HEK293 cells, which served as a noncancerous model. Previously synthesized vanadium complexes (VCs) with 5‐Br and 3‐OMe functional groups were used as treatments in vitro. A cell proliferation assay was used to assess HEK293 growth inhibition after treatment with each VC. Results showed 55.2% (p<0.05) and 24.6% (p<0.05) growth inhibition for 0.5mM 5‐Br and 3‐OMe VCs, respectively, which is comparable to previously presented results with the HT‐29 cells. An intracellular ROS generation assay was used with both HT‐29 and HEK293 cell lines after being treated with 0.25 mM 5‐Br and 3‐OMe VCs for 4, 24, 48, and 72‐hour incubation periods. For the HT‐29 cells, there were no significant increases in ROS generation after 4, 24, or 48‐hour incubation periods with either treatment. After 72 hours, however, the 5‐Br‐VO2 yielded a 51.3% (p<0.05) increase in intracellular ROS generation compared to the ligand control, and the 3‐OMe‐VO2 yielded a 46.0% (p<0.05) increase. For the HEK293 cells, there was likewise no significant increase in ROS generation after 4‐hour incubation periods, but there was a 44.1% (p<0.05) increase in ROS generation after 24‐hour incubation with the 5‐Br‐VO2 alone. There were no significant increases in ROS generation after the 24‐hour incubation period in the HEK293 cells. Finally, a caspase 3/7 apoptosis detection assay was also used to determine caspase pathway activation. Compared to the cisplatin positive control, the 5‐Br‐VO2 treatment yielded no measurable caspase activation in the HT‐29 cells after 16 and 24 hours of incubation.

Conclusion

We found that VCs were able to effectively inhibit HT‐29 growth through likely intracellular generation of ROS, rather than through caspase activation of apoptosis.

Support or Funding Information

Hewlett‐Mellon Fund for Faculty Development at Albion College
Intermittent Hypoxia Decreases Baroreflex Sensitivity in Healthy Humans
Authors: Scruggs Zachariah M , Newhouse Lauren P , Joyner Michael J , Curry Timothy B , Limberg Jacqueline K
Published: November 2023

Background

Patients with sleep apnea experience repeated, intermittent hypoxic exposures during sleep and are at increased risk of developing hypertension. The mechanisms that contribute to high blood pressure in these patients are not completely understood. In this context, we sought to examine the effect of intermittent hypoxic exposures on baroreflex sensitivity and blood pressure. We hypothesized 3‐hours of intermittent hypoxia would result in a decrease in baroreflex sensitivity in healthy adults. We further hypothesized any decrease baroreflex sensitivity would be related to an increase in blood pressure following intermittent hypoxia.

Methods

Six (5M/1F) young healthy, non‐obese, subjects without sleep apnea completed 3‐hours of intermittent hypoxia. Intermittent hypoxia consisted of 25‐second exposures to hypoxic gas (0.05 FiO2) separated by 2 minutes of room air breathing, resulting in 25‐hypoxic events per hour in which oxygen saturation (SpO2) was significantly reduced (Normoxia: 97%, Hypoxia: 89%, p<0.01). Beat‐to‐beat heart rate (electrocardiogram) and blood pressure (finger photoplethysmography) were measured continuously. Spontaneous cardiac baroreflex sensitivity was determined using the sequence method (WinCPRS) and data are reported from 10‐minutes of quiet, normoxic rest before and after the intermittent hypoxia protocol.

Results

We observed a significant reduction in spontaneous cardiac baroreflex sensitivity to falling pressures (“down‐down”) following intermittent hypoxia (Control=22±2, IH=15±2 ms/mmHg; p=0.02). No change in baroreflex sensitivity to rising pressures (“up‐up”) was observed (Control=25±2, IH=20±6 ms/mmHg; p=0.41). A relationship between baroreflex sensitivity and mean arterial blood pressure was observed, such that a reduction in baroreflex sensitivity tended to be related to an increase in blood pressure (“down‐down”: R=−0.45, p=0.14; “up‐up”: R=−0.54, p=0.07).

Conclusions

Intermittent hypoxia results in a decrease in baroreflex sensitivity, which is related to an increase in blood pressure. These data may have important implications for mechanisms contributing to the risk of hypertension in individuals with sleep apnea.

Support or Funding Information

DK90541 (MJJ), HL120570 (JKL)
Localization of corticotropin releasing factor receptor 2 (CRFR2) in the nucleus tractus solitarii (nTS)
Authors: Ruyle Brian C. , Callanan Gabrielle F. , Haney Megan M. , Coldren K. Max , Heesch Cheryl M. , Hasser Eileen M.
Published: November 2023

The paraventricular nucleus of the hypothalamus (PVN) is required for full expression of cardiorespiratory responses evoked by peripheral chemoreflex stimulation. However, the efferent pathways from the PVN that contribute to chemoreflex responses are not fully understood. We have previously shown that hypoxia activates (Fos‐immunoreactivity; IR) PVN neurons that project to the nTS, and a large proportion of these neurons are CRH‐IR. Activation of CRF2 receptors (CRFR2) in the nTS elicits cardiovascular responses, and thus we considered that CRH PVN neurons may influence nTS neuronal activity and cardiorespiratory effects of chemoreflex activation through binding of CRH to CRFR2s. Immunohistochemistry was performed to determine the distribution of CRFR2 in the nTS. CRFR2‐IR was not identified in neurons or glia but was localized in fibers throughout the caudal, postremal and rostral nTS. CRFR2 formed close appositions on dendrites (displaying MAP2‐IR) of nTS catecholaminergic (tyrosine hydroxylase; TH‐IR) neurons and a portion of CRFR2 was colocalized with the synaptic vesicle protein Synaptophysin (Syn). An AAV viral vector expressing mCherry under the promoter CAMKII was microinjected into the PVN and mCherry containing fibers in nTS were evaluated. Terminals in the nTS originating from the PVN (mCherry) also exhibited colabeling with Syn and appeared to make close appositions on TH‐IR nTS neurons. Interestingly, CRFR2‐labeled fibers were colocalized extensively with oxytocin‐IR that also exhibited Syn‐IR. These results suggest that CRH, likely from the PVN, may bind to presynaptic CRFR2s located on oxytocin‐containing fibers to modulate the activity of catecholaminergic nTS neurons and influence cardiorespiratory regulation.

Support or Funding Information

NIH RO1 HL98602
Higher Cell Growth/Viability Accompanied by Reduced Myosin Heavy Chain Expression in Skeletal Muscle Cells Derived from Severely Obese Type 2 Diabetic Humans
Authors: Turner Kristen D , Zou Kai , Hinkley Matthew , Park Sanghee , Zheng Donghai , Houmard Joseph
Published: November 2023

Skeletal muscle metabolism is markedly impaired in a state of severe obesity (BMI ≥ 40 kg/m2) and oftentimes worsened with Type 2 Diabetes. Such impairments have been demonstrated through our previous findings in vitro, in which both glycolytic and oxidative metabolism are reduced in skeletal muscle from severely obese (SO) as well as severely obese type 2 diabetic individuals (T2D). These data suggest that such metabolic differences observed may be intrinsic to skeletal muscle. However, we have not yet provided empirical evidence to show whether these differences could be due to other physical properties associated with these muscle cells (i.e., viability, morphology, muscle cell machinery). Thus, the purpose of this study was to examine the growth/viability of human skeletal muscle cells (HSkMCs) derived from SO and T2D individuals in comparison to lean individuals. This study also determined whether mature myotubes exhibited differences in myosin heavy chain (MYHC) expression among groups. We hypothesized that skeletal muscle cells derived from T2D donors would exhibit lower cell viability and MYHC expression in comparison to their lean and SO counterparts. Interestingly, colorimetric metabolic activity assays revealed myoblast cell viability to be significantly higher in myoblasts derived from T2D donors at 24, 48 and 72 hours post‐plating. However, immunocytochemical staining of MYHC (performed on days 6–7 of differentiation) illustrate considerably less MYHC expression in mature myotubes derived from T2D donors. These findings demonstrate that severely obese type 2 diabetic individuals exhibit critical defects in both structural and functional properties of skeletal muscle that are retained at the cellular level, and that such differences may not always be apparent with severe obesity alone.

Support or Funding Information

This study was supported by the National Institute of Health (DK 56112)
The Enhanced CO2 Chemoreflex is Associated with Increased Number of CO2‐activated Orexin Neurons in the Lateral Hypothalamus in Spontaneously Hypertensive Rats (SHRs)
Authors: Li Aihua , Nattie Eugene , Roy Sarah
Published: November 2023

Activation of central chemoreceptors by CO2 increases arterial blood pressure (ABP), sympathetic nerve activity (SNA), and breathing. Experiments using SHRs, a well‐established neurogenic hypertensive model, have shown that the high ABP is associated with elevated SNA and peripheral chemoreflexes, and an impaired baroreflex. We have showed that: 1) SHRs have an age‐dependent increase in CO2 chemoreflex and ABP; 2) the increased CO2 chemoreflex and ABP are measureable in juveniles (P28–50); 3) the enhanced CO2 chemoreflex persists while peripheral chemoreceptors were inhibited by hyperoxia indicating an important role for central chemoreflex in neurogenic hypertension; and 4) blocking orexin receptors with a dual orexin receptor antagonist, Almorexant (Almxt), can normalize the enhanced CO2 chemoreflex and significantly lower high ABP in SHRs. In this study, we evaluated CO2‐activated c‐fos expression in orexin neurons in both hypertensive (SHRs) and normotensive control (WKY) rats, and hypothesized that the enhanced CO2 chemoreflex observed in SHRs is associated with increased number of CO2‐activated c‐fos expressing orexin neurons in the lateral hypothalamic area (LHA). The rats were exposed to 7%CO2 for 1.5hr, and sections of LHA were double stained for c‐fos and orexin‐A. The Stereo Investigator System was used for the cell counts. The results show that in the LHA, SHRs have: 1) more orexin‐containing neurons (~28%), similar as some recent reports, and 2) more CO2‐induced c‐fos and orexin double‐expressing neurons (~71%) than that of age‐matched normotensive WKY rats. We conclude that the enhanced CO2 chemoreflex in SHRs is associated with the overactive orexin system, and modulation of this overactive orexin system and CO2 chemoreflex could be beneficial in treating neurogenic hypertension.

Support or Funding Information

NIH HL 28066
Characteristics in the Vulnerability of the Respiratory Neural Control System During a Critical Window of Postnatal Development
Authors: MacFarlane Peter Mathew , Mayer Catherine Ann
Published: November 2023

Prior studies have demonstrated the existence of a critical window of neurochemical expression that occurs in the brainstem at approximately 12 days of postnatal (P12) age in the rat. The critical window is characterized by changes in the expression of various neurotransmitters, receptors, trophic factors, and metabolic enzymes in various brainstem respiratory control regions. Presumably these changes represent important neurodevelopmental events necessary for appropriate maturation of the cardio‐respiratory control system. However, studies from our lab have begun to demonstrate the critical window is a period of development during which the respiratory control system exhibits a heightened vulnerability to environmental challenges. For example, five days of sustained hypoxia exposure (SH: 11% O2) between P11–15 days, which encompasses the P12 critical window, abolished the ventilatory response to acute hypoxia (HVR (delta baseline): 0.63 ± 0.31 ml/g/min) compared to age‐matched rats raised in normoxia (0.12 ± 0.45 ml/g/min). The absence of a HVR in these rats was associated with an unexpected and substantial degree of mortality that occurred ~3 days later. These effects were unique to the critical window as neither younger (P1–5) or older (P21–25) rats were affected by the same exposure to SH. We have also shown the critical window appears to be an important stage of brainstem neurodevelopment because it is represented by a transient surge in constitutive expression of brainstem mRNA for NeuroD1 and CyclinB2. However, SH between P11–15 reduced their expression (NeuroD1: 0.66 ± 0.11; CyclinB2: 0.74 ± 0.14 fold‐change from normoxia control rats), indicating a major and potentially fatal disturbance in brainstem mechanisms of neurodevelopment. The critical window of development was also characterized by a transient increase in the constitutive expression of brainstem TNFα mRNA, which was further increased by SH exposure (1.47 ± 0.09 fold‐change from control). Further, SH also decreased serotonin (5‐HT) levels specifically within the dorsal motor nucleus of the vagus (DMNV) and nucleus of the solitary tract (nTS). We speculate that SH exposure interferes with key neurodevelopmental events, ultimately leading to a lethal disturbance in brainstem neurochemistry within distinct (cardio)–respiratory control regions and may involve a mechanism associated with an aberrant cytokine response. These data could contribute to our understanding of the pathways involved in the brainstem neurochemical abnormalities observed in Sudden Infant Death Syndrome (SIDS).

Support or Funding Information

Department of Pediatrics, Rainbow Babies & Children's Hospital, Case Western Reserve University
Pros and Cons of Active Learning
Authors: Chakraborty Tandra R.
Published: November 2023

Teaching is an art and can be done in various ways. I am a strong believer of traditional teaching technique (class lecture and Socrates method of testing student learning) but integrating active, cooperative and collaborative learning alongside makes the learning experience of students fun and memorable. Some of the active learning strategies that I find most beneficial are, formal debate, role playing (dramatic presentation, using technology), group work, clarification pauses, and documentary movies related to the topic of interest, peer survey's and flip the classroom. Although the benefits of these teaching strategies are many there are some difficulties that come with it like time and material covered in class is less, lack of individual accountability, difficulty in grading, designing of activity that fits the course.
Body Mass Estimation from the Knee: Forensic Applications
Authors: Squyres Nicole , Ruff Christopher
Published: November 2023

The estimation of living body mass from skeletal material can be a useful component of a forensic profile. Previously, forensic anthropologists have generally used body mass estimation equations derived either from the femoral head or from a combination of bi‐iliac breadth and stature. However, these equations were originally generated for use in archaeological or paleontological contexts, and therefore may not perform as well in modern sedentary populations. In particular, these equations may significantly underestimate body mass in overweight and obese individuals, which increasingly account for a greater percentage of living populations.

Here, we present new body mass estimation equations derived from measurements of the knee in a modern sample of known body mass. This reference sample consisted of 110 living subjects who participated in the Baltimore Longitudinal Study of Aging. Mediolateral breadth measurements of the distal femur and proximal tibia were taken from anteroposterior radiographs of the knee, and used with recorded body weight to develop prediction equations. We compare the results from equations derived from the entire reference sample with equations derived from a sample that excludes obese individuals (BMI>30). We also compare equations derived from youngest recorded body mass with equations derived from older body mass recorded at the time of x‐ray.

R square, % standard error of the estimate, and % prediction errors are minimized in equations that use youngest recorded body mass and exclude obese individuals. Although body mass for obese individuals is underestimated by all equations, it is best approximated by equations derived from the older recorded body mass that include obese individuals. This type of equation also provides very good estimates of body mass for overweight individuals (25<BMI<30). All equations overestimate body mass to some extent in normal weight individuals, but equations derived from young adult body mass excluding obese individuals perform best in this group. Comparison with other studies suggests that certain of these equations may better approximate body mass in obese and overweight individuals than previously used equations.

Our results indicate that, due to increasing rates of obesity in modern societies, body mass estimation equations designed for paleontological use should be applied with caution in the forensic context. Equations derived from modern samples with a greater proportion of overweight and obese individuals will perform better in such cases.
Using Novel Methods to Visualize Jaw Muscle Biomechanics and its Significance for the Evolution of the Avian Feeding Apparatus
Authors: Spates Anthony T , Cost Ian N , Sellers Kaleb C , Middleton Kevin M , Holliday Casey
Published: November 2023

The avian feeding apparatus is a derived system of bony linkages spanned by numerous jaw muscles in a complex 3D environment. Tracking the evolution of jaw muscles among different clades of birds and their extinct dinosaur relatives remains challenging because of this complexity. Here we introduce novel means of mapping 3D jaw muscle resultants in ternary space to seek patterns in the form, function and evolution of avian head and determine the origins of cranial kinesis. For this pilot project, we built 3D biomechanical models of four taxa of birds (ostrich, chicken, hawk, and parrot) and their extinct relative Tyrannosaurus thus spanning a range of cranial morphology, feeding function, and phylogeny. Specimens were microCT‐scanned using normal and contrast enhanced methods, muscles were dissected and physiological cross sectional areas were measured to determine muscle forces. Muscles were then reconstructed in the models and 3D lever analysis was undertaken using BoneLoad and other computational tools to determine 3D muscle resultants, bite forces and joint forces. 3D muscle resultants were then plotted in a ternary space to illustrate the variation in positions and magnitudes of the different jaw musculature. Our findings show many new patterns in avian jaw muscle anatomy and function including: 1) evolutionary convergence in orientation, position, and function of non‐homologous temporal muscles between ostrich and other taxa, 2) Chicken jaw muscles occupy a small, primarily vertically‐oriented portion of the available morphospace whereas the muscles of the parrot occupy a far broader and diverse morphospace reflecting their highly dexterous feeding apparatus, and 3) Protractor muscle resultants vary considerably among the taxa sampled suggesting different capacities for palatal mobility and cranial kinesis. These findings highlight the comparative power of these new methods for visualizing jaw muscle functional morphology in birds and other vertebrates and will offer significant new direction in comparative morphometric, biomechanical and evolutionary analyses.

Support or Funding Information

This work was funded by NSF IOS 1457319, the University of Missouri Express Program and the University of Missouri Honors Program.
The chromatin remodeling complex SWI/SNF is a master regulator of meiotic splicing in Saccharomyces cerevisiae
Authors: Venkataramanan Srivats , Douglass Stephen , Galivanche Anoop Raj , Johnson Tracy
Published: November 2023

Despite its relatively streamlined genome, there are important examples of regulated RNA splicing in Saccharomyces cerevisiae. One of the most striking is the splicing of meiotic transcripts. At the onset of meiosis, Saccharomyces cerevisiae, like other eukaryotes, undergoes a dramatic reprogramming of gene expression. This includes regulated transcription and splicing of a number of meiosis‐specific transcripts. Splicing of a subset of these is dependent upon the meiosis‐specific splicing activator Mer1. Here we show a crucial role for the chromatin remodeling complex Swi/Snf in meiotic regulation of splicing. We find that the complex affects meiotic splicing in multiple ways. First, meiosis‐specific downregulation of Snf2, the ATPase subunit of Swi/Snf, regulates the subsequent downregulation of ribosomal protein genes (RPGs), RPGs are enriched in introns, and are highly transcribed. Their downregulation leads to the redistribution of spliceosomes from this abundant class of intron‐containing RNAs to Mer1‐regulated transcripts, which otherwise have poor affinity for the spliceosome. Secondly, expression of Mer1 itself is absolutely dependent on Swi/Snf—Snf2 is poised at the Mer1 promoter, and the timing of Snf2 downregulation allows elegant coordination of these mechanisms. This precisely timed meiotic regulation of Snf2 involves co‐ordination of regulated transcription, translation, protein acetylation and turnover. Hence, the Swi/Snf complex directs the regulated splicing of meiotic genes, establishing it as a master regulator of meiotic splicing in Saccharomyces cerevisiae. Moreover, meiosis serves as an elegant example of a poorly understood, but undoubtedly important general regulatory mechanism employed by cells, whereby redistribution of limiting gene‐regulation machineries to different classes of targets serves as an adaptive strategy in response to environmental changes.

Support or Funding Information

NIH GM05474

NIH P50 GM085764

Whitcome Pre‐Doctoral Fellowship in Molecular Biology.
Examination of the Mechanism of Intersubunit Communication in Pyruvate Carboxylase Using Mixed Hybrid Tetramers
Authors: Westerhold Lauren E. , Zeczycki Tonya N.
Published: November 2023

Pyruvate carboxylase (PC) is a biotin‐dependent, homotetrameric enzyme that is responsible for the MgATP‐dependent catalytic conversion of pyruvate to oxaloacetate in the presence of the essential allosteric activator, acetyl‐CoA. PC is a critical anaplerotic enzyme in glucose metabolism, serving to replenish oxaloacetate consumed by the TCA cycle. Increased production of acetyl‐CoA, due to high lipid supply, has been shown to upregulate PC activity in the liver, contributing to the development of obesity‐induced insulin resistance and Type 2 Diabetes. Previous crystallographic studies have identified four distinct domains that comprise the PC monomer, namely the biotin carboxylase (BC), biotin carboxyl carrier (BCCP), carboxyl transferase (CT), and allosteric domains. Each domain has been functionally characterized via steady‐state kinetics and site‐directed mutagenic studies of catalytically relevant residues. The tetramer is a dimer of dimers, whereby each face is composed of a dimer of monomers arranged antiparallel to each other.

Pyruvate binding has been shown to trigger the movement of the BCCP domain to an opposing polypeptide chain, though it is not known whether this is facilitated through an inter‐or intramolecular “signal.” That is, does pyruvate binding on the same monomer or on the opposing polypeptide chain facilitate the intermolecular BCCP domain translocation? To determine whether BCCP domain movements are governed by intermolecular or intramolecular mechanisms, we have generated PC hybrid tetramers (HTs) consisting of two strategically chosen mutants: one with an inactive BC domain and wild‐type CT domain (E218A); and the other with a wild‐type BC domain and a mutant CT domain (T882S) exhibiting a 62‐fold decrease in kcat and 3‐fold increase in Km, pyruvate as compared to wild‐type. Steady‐state kinetics of the full‐forward reaction catalyzed by the T882S:E218A(1:1) HT demonstrate that the pyruvate concentration needed to facilitate BCCP translocation is significantly higher than that required for the carboxylation reaction. More importantly, our results indicate that the occupancy of the CT domain by pyruvate on an opposing polypeptide chain “triggers” the translocation of the BCCP domain from the BC domain on a monomer to the opposing CT domain. Further study of additional molecular interactions within the tetrameric structure, such as BC—BC domain interactions between faces of the enzyme, will be used to elucidate the nature of the mechanism by which PC controls BCCP translocation and the coordination of catalysis between two spatially distinct active sites.
Control of Enzyme function through loop dynamics
Authors: Chi Celestine N. , Vogeli Beat , Bibow Stefan
Published: November 2023

Allosteric regulation has been revealed as an essential property of all proteins. Dynamic shift of structural equilibrium at one site can have structural and functional effect at other sites. However for small, single domain proteins these phenomena is difficult to access, albeit quantify easily. One way of shifting structural equilibrium distribution is by changing the the dynamics of flexible loops by amino acid substitution because large changes maybe achieved with minimal modification of protein. A ligand‐selective change of the binding affinity to the active‐site of the enzyme cyclophlin by tuning the dynamics of a highly flexible loop is presented.

This system may serve as a platform to study the effects of various timescales of loop motion on protein function turned by mutations and opens the possibility for elucidating the role of time‐scale dependent allostery

Support or Funding Information

This work was support in part by the Swedish research council Wenner‐Gren Stiftelsern WG‐13, Postdoctoral fellowship to C. N. Chi, the Swiss National Science Foundation with Grant 140214 and ETH Research Grant ETH‐04 13‐1 to B. Vögeli.
Unexpected pH‐dependent DNA Binding of CooA, a CO‐Sensing Transcription Factor from Rhodospirillum rubrum
Authors: Weaver Brian R. , DeVries Rachael M. , Gunter Amy J. , Clark Robert W.
Published: November 2023

CooA, a CO‐sensing heme protein, acts as a transcriptional activator of CO metabolizing proteins in Rhodospirillum rubrum and other bacteria through sequence‐specific DNA binding. Prior research has indicated that a reduced iron center and CO gas were necessary for CooA to achieve its active conformation and bind DNA. To determine if other reaction conditions facilitate CooA activation, the role of pH on CooA DNA binding was tested. Using a fluorescence anisotropy assay, Fe(III) CooA was observed to bind DNA without the presence of gas under acidic conditions (pH < 7). Specifically, DNA binding was quantified from pH 4 – 10, and optimal binding was observed at approximately pH 4. These results are discussed in light of the normal CO‐dependent activation mechanism of CooA proteins.
Regulation of N‐myristoyltransferase by Akt/PKB Mediated Phosphorylation
Authors: Kumar Sujeet , Sharma Rajendra K.
Published: November 2023

Post‐translational modifications of proteins create highly dynamic relay system that regulates the core dynamic signalling pathways in response to the alterations in the cellular microenvironment. N‐myristoyltransferase (NMT) has emerged as a central node upstream of diverse signalling proteins regulating cellular survival. NMT modifies proteins in both co‐and post‐translational manner and modulates functioning of the signalling proteins by addition of the myristoyl moiety to an exposed N‐terminal glycine. The sequence feature of NMT reflects that it harbours an Akt/PKB kinase recognition motif within the aD‐ aE loop region. The in vitro kinase assay on the synthetic peptides corresponding to the aD‐aE loop followed by MALDI‐MS analysis established that the sequence motif is a valid substrate for the Akt/PKB kinase. The MS‐MS analysis of the phosphorylated peptide identified Thr319 within aD‐ aE loop region as the site of phosphorylation. To delineate the effects of phosphorylation, phosphomimetic mutant (Thr319Glu) and a null‐mutant (Thr319Ala) were engineered onto the catalytic domain the NMT. Enzymatic assays of the purified recombinant enzymes reflected a drastic loss in activity by mutations at the site Thr319. The analysis of NMT crystal structures shows that this loop region undergoes a conformational change upon binding of the co‐substrate myristoyl‐CoA (MYA). The binding of peptide substrate follows the MYA binding event and Thr319 in the aD‐ aE loop region lies in proximity to the peptide‐substrate binding site. In summary, we have established the site of phosphorylation and the regulation of NMT by the kinase Akt/PKB. These findings indicate that Akt/PKB kinase restricts NMT activity by modulating the peptide substrate affinity presumably by changing in the interaction network around the substrate‐binding site. Delineating of the functional consequences of this novel phosphorylation event is expected to improve our understanding of NMT regulations in diversified cellular needs.

Support or Funding Information

This work was supported by the Canadian Breast Cancer Foundation‐Prairies/NWT operating grant to RKS (Grant number ‐ 412572).
Modification of the flexible loop of tyrosine hydroxylase that determines substrate specificity
Authors: Daubner Susan Colette , Rios Socorro
Published: November 2023

Tyrosine hydroxylase (TyrH) is one of three aromatic amino acid hydroxylases, all of which evolved from an ancient phenylalanine hydroxylase (PheH). Modern eukaryotic PheH is unable to convert tyrosine to DOPA. Tyrosine hydroxylase is able to hydroxylate phenylalanine and tyrosine. One residue in particular allows the new activity, the aspartate at position 425 in TyrH (the homologous residue in PheH is valine 379)1. Aspartate 425 lies in a flexible loop situated over the enzyme active site. Previous work showed that TyrHD425E has only 10% of the activity of wild‐type TyrH, and that hydrophobic residues substituted in position 425 abolished TyrH activity. To understand better the flexible loop's structure and role in substrate specificity, we have made shape‐altering changes to TyrHD425E, deleting the adjacent glutamine residues. These include TyrHdeltaQ424D425E (Q424 removed), TyrHD425E deltaQ426 (Q426 removed), and TyrHdeltaQ424D425EdeltaQ426 (both glutamines removed). Attempts to grow these variants in E. coli have varied in success. As assessed by SDS gel electrophoresis of crude lysates, TyrHD425EdeltaQ426 is expressed better than TyrHdeltaQ424D425E. Both enzymes are expressed comparably to wild‐type enzyme, but the variant lacking both glutamines is not expressed under routine growth conditions. The partially purified enzymes have been tested for DOPA‐synthesizing activity. These preliminary measurements suggest that both variants that lack one glutamine residue are able to convert tyrosine to DOPA at faster rates than TyrHD425E.
Organic synthesis of novel phosphatidylinositol 3‐kinase (PI3K) inhibitors activated by prostate specific antigen (PSA) for the treatment of androgen‐independent prostate cancer
Authors: Nelson Ronald Alvin
Published: November 2023

The PI3K/Akt pathway is one of the most frequently dysregulated signaling pathways in cancer and remains a crucial target in drug development. Current therapeutic drugs are potent, but often lack the cellular specificity needed to lower toxicity effects. Here we show the design and synthesis of two types of novel PSA‐activated PI3K inhibitors for the treatment of androgen‐independent prostate cancer. Both literature and predictive molecular modeling studies suggest PI3K enzyme inhibition in the nanomolar/picomolar range. Type 1 PI3K Inhibitors contain : (1) a pyrimidine or triazine core, (2) a secondary amine ‐ usually a morpholine ring, and (3) an aromatic/heteroaromatic group capable of hydrogen bonding. Type 2 PI3K Inhibitors contain these same three moieties but with added electrophilic functional groups or electron withdrawing groups (EWG) on the secondary amine and the aromatic/heteroaromatic group. These functional groups will allow reversible covalent bond forming reactions to occur with nucleophilic amino acids in the active site of PI3K ‐ an approach that has not been attempted. Reversible covalent bond forming reactions are hypothesized to allow the inhibitor to stay in the active site longer and thus enhance it's potency towards the targeted enzyme. Additionally, the attachment of a peptide chain, with the embedded amino acid sequence HSSKLQ recognized and cleaved by prostate specific antigen (PSA), to both Type 1 and Type 2 PI3K inhibitors is hypothesized to significantly improve specificity towards prostate cells (based off our previously published work) and subsequently: (1) prevent further cellular proliferation of prostate cancer cells, (2) lower toxicity effects, and (3) induce apoptosis through Caspase activity. Testing our active Type 1 PI3K inhibitors via western blot analysis against the current clinical trial PI3K inhibitor ZSTK474 showed 10 fold improved inhibition. Predictive trends for our Type 2 PI3K inhibitors, based off of previous data, suggests 100 fold (or greater) improved inhibition when compared to ZSTK474.

Support or Funding Information

The author would like to thank Dr. Fred Salsbury for the molecular modeling and computational work, Dr. George Kulik's lab for previous drug screening data, Dr. Welker's Lab for prior organic synthesis developments, and Wake Forest University's Chemistry Department for providing lab resources and equipment. The author would also like to thank Wake Forest University's Center of Molecular Communication and Signaling for financially supporting this project.
In silico Selective Screening for Inhibitors of ABC Transporters that Minimizes Off Target Toxicity
Authors: McCormick James W. , Follit Courtney A. , Vogel Pia D. , Wise John G.
Published: November 2023

The ATP‐binding cassette (ABC) class of transporters is involved in many diseases. Chemotherapy resistant, recurrent cancers commonly express ABCB1 P‐glycoprotein transporter (P‐gp) and ABCG2 Breast Cancer Resistance Protein (BCRP). At high levels of expression, both transporters lower the intracellular concentration of chemotherapeutics to a subtherapeutic level. The prokaryotic transporter MsbA is an essential ABC lipid A flippase that is homologous to the human multidrug resistance proteins. MsbA serves both as a drug target as well as an indicator of possible off target interaction of P‐gp/BCRP inhibitors due to the similar structure and ease of assays in bacterial model systems. Currently used methods of computational screening for inhibitors lack discrimination against off target interactions and are dependent on the quality of the starting structure. These problems are compounded with ABC transporters since inhibitors have historically been substrates of the transporters and often are substrates for cytochrome P450 3A4, which is heavily involved in the oxidation and metabolism of many drugs. These problems, and the lack of any high resolution crystal structure for the human proteins, has made finding ABC transporter inhibitors suitable for human use elusive.

Previous work identified multiple structures of P‐gp that we used for computational screening for potential inhibitors. To prevent inherent biases from single structures, simulations of a catalytic cycle using targeted molecular dynamics was performed (Wise 2012 Biochemistry 51(25), 5125–5141; McCormick et al. 2015 Biochemistry 54(28), 4374–4390.). Molecular dynamics simulations of MsbA and Cyt P450 3A4 were also used to generate screening targets. Targeting the ATP binding domains and drug binding domains of P‐gp, and related domains of MsbA and the active site of P450 allowed for selection of hit compounds that are specific to one region of the protein of interest and avoid off site interactions. To limit computational time, iterative screening on similarity subsets of the ZINC database was performed, with subsequence screens based on the diversity of hits found in initial screens. Top hit compounds were purchased and assayed using biochemical and cell culture methods as described in Brewer et al. 2014 Molecular pharmacology 86(6), 716–726 and Follit et al 2015 Pharmacology Research & Perspectives 3(5), e00170. The correlation between computational screening methods and results from biochemical and cell culture assays for inhibition of P‐glycoproteins is described.

Support or Funding Information

This work is supported by NIH NIGMS [Grants R15‐GM094771‐01A1 and R15‐GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzann Ruff of Dallas, Texas.
Treatment with diet or insulin induces a different placental Ceramide expression during gestational diabetes mellitus (GDM)
Authors: Mejia Camilo A. , Monson Troy , Jordan Clinton , Bikman Benjamin T. , Reynolds Paul R. , Arroyo Juan A.
Published: November 2023

Gestational diabetes mellitus (GDM) is associated with a number of factors that prevent the placenta from transporting necessary nutrients and oxygen important for fetal development. Ceramides are a family of lipids that consist of sphingosine covalently linked to a fatty acid that is known to be associated with the development of diabetes and the induction of cell apoptosis. Our objective was to determine levels of ceramide and apoptosis during gestational diabetes treated with insulin or diet. Placental biopsies and paraffin embedded placental tissues slides for GDM‐I (gestational diabetes mellitus treated with insulin), GDM‐D (gestational diabetes mellitus treated with diet), and term controls were obtained. Immunohistochemistry was preform for ceramide localization in the placenta. Immunoblot was done for serine palmitoyltransferase (SPT; enzyme involve in ceramide production), active caspase 3 and XIAP protein (inhibitor of caspase activation). There were no significant differences in maternal age, gestational weeks and fetal weight between control and GDM pregnancies. Immunohistochemistry showed an increased in ceramide in the villi trophoblast of the placenta during GDM‐I but not in the GDM‐D tissues. Similarly, an upregulation for nuclear SPT expression, the rate‐limiting enzyme in de novo ceramide biosynthesis, was present only in the GDM‐I placenta (3.4‐fold; p<0.05) as compared to controls. In contrast, no significant differences were observed for cytosolic SPT expression between control and GDM tissues. There was a similar upregulation for active caspase 3 (1.2‐fold; p<0.05) in the placenta of both GDM‐I and GDM‐D when compared to control placental tissue. Interestingly no significant differences were observed for cytosolic XIAP between samples, while there was a significant decrease for nuclear XIAP only (1.7‐fold; p<0.05) in the GDM‐I placenta as compared to controls. Our findings confirm the presence of ceramide in the human placenta of control and GDM patients. Furthermore our results demonstrated that ceramide is increased in the placental trophoblast during insulin treatment and that this increased correlate with the increased caspase activation and decreased XIAP observed during this treatment. Our results also suggest that the increased activation of placental caspase 3 during GDM‐D is occurring in a ceramide independent manner. Treatment variation between diet and insulin tissues suggest that an equally effective but different mechanism is exerted in the placenta leading to the alleviating of the symptoms/consequences present during GDM in the placenta.
Proximate composition and nutritional profile of Rhynchophorus palmarum
Authors: Okunowo Wahab Oluwanisola , Olagboye Abimbola Mary
Published: November 2023

The larva of the insect Rhynchophorus palmarum is one of the species of insects consumed by the rural people in Nigeria and little or scarce nutritional data exist. The proximate composition of the larva was determined by standard analytical methods. The amino acid composition was determined using an Amino Acid Analyzer, the fatty acid profile was determined with the aid of a Gas Chromotography‐Mass Spectrophotometer, the vitamins and mineral contents were analyzed by High Pressure Liquid Chromatography and Atomic Absorption Spectrophotometer respectively. Results showed that the proximate composition in percentage for crude protein, crude lipid, crude carbohydrate, crude fiber and ash content were 24.43 ± 1.30, 15.36 ± 0.82, 2.27 ± 0.28 and 1.00 ± 0.19 respectively. The non essential amino acids were highest proline (14.0 g/100g protein) and glutamic acid (13.0 g/100g protein), and the essential amino acids were highest Leucine (6.74 g/100g protein) and Lysine (5.83 g/100g protein). The major fatty acids were Heptadecanoic acid (43.4%), 6‐Octadecanoic acid (31.06%) and cis‐13‐Octadecanoic acid (20.24%). The unsaturated/saturated fatty acid ratio was 1.11. The major water soluble vitamins were Niacin (35,166mg/100g) and Vitamin C (11.14 mg/100g), and fat soluble vitamins were Vitamins K (165.46 mg/100g) and E (125.18 mg/100g). The results also indicate that the larva was rich in Iron (350.3 mg/kg), calcium (324.8 mg/kg), potassium (249.3 mg/kg) and sodium (160.7 mg/kg). Lead, Cadmium and chromium were not detected. This study shows that R. palmarum larva could be used as human dietary supplement to balance diet.