bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Morphological Features of Fibrillar Collagen Indicative of Tumor Progression in Bladder Cancer
Authors: Borhani Soheila , Macias Virgilia , Kajdacsy‐Balla Andre
Published: May 2022

Objective

Bladder cancer is the most common malignancy involving the urinary system. As with all types of cancer, accurate monitoring of stage progression is critical in determining proper treatment strategies and predicting clinical outcomes. Collagen fibers are a major component of tumor microenvironment, and their post‐translational modifications during the course of tumor progression are well‐documented in a number of malignancies including bladder cancer. In the present feasibility study we sought to determine whether quantitative morphological features extracted from collagen fibers can be used to distinguish between urothelial carcinoma tumors pre‐muscle‐invasion (stage pT1) and post‐muscle‐invasion (stage pT2).

Methods

PicroSirius Red (PSR) staining was used to detect fibrillar collagen in 8 tissue microarray (TMA) cores acquired from Biomax (4 stage pT1, 4 stage pT2). We applied auto‐fluorescence imaging techniques to detect the PSR signal, and employed the CT‐Fire freeware to automatically extract individual collagen fibers. Five morphological features (including length, width, straightness, orientation, and anisotropy) were computed for each detected fiber. Individual features were then combined within each slide to form five corresponding histograms per TMA core. Finally, for each histogram we computed two measures of central tendency (i.e., mean and median), two measures of spread (i.e., range and standard deviation), and two measures of symmetry (i.e., skewness and kurtosis).

Results

To identify any statistically‐significant differences between the pT1 and pT2 groups, we employed the student’s t‐test (run in Python 3.8.5) to compare the two groups with respect to each of the five morphological features and across each of the six distributional measures mentioned in the Methods section (30 comparisons in total). Of these 30 comparisons, all but one (i.e., median) of the six straightness measures resulted in a p‐value < 0.05. Further, each of the two measures of spread (i.e., range and standard deviation) of straightness returned a p‐value < 0.001. We are currently working on expanding the size of our dataset to further validate these results.

Conclusion

Our preliminary findings are in line with the hypothesis that collagen remodeling is strongly associated with muscle‐invasive bladder cancer, and suggest that quantitative features measuring the straightness of individual fibers may be used to improve tumor staging. This type of quantitative approach is particularly helpful in distinguishing between stage pT1 and stage pT2 of bladder cancer, a task which can be challenging at times even for experienced pathologists.
Increased phosphate absorption in mice lacking NHE3 in intestinal epithelial cells
Authors: Thomas Linto , Xue Jianxiang , Murali Sathish K. , Levy Moshe , Fenton Robert A. , Dominguez Rieg Jessica A. , Rieg Timo
Published: May 2022

The kidneys play a major role in maintaining phosphate (Pi) homeostasis. Patients in later stages of CKD develop hyperphosphatemia. One novel treatment option is tenapanor, an intestinal‐specific NHE3 inhibitor. To gain mechanistic insight into the role of intestinal NHE3 in Pi homeostasis, we studied tamoxifen (Tam) ‐inducible intestinal epithelial cell‐specific NHE3 knockout (NHE3IEC‐KO) mice with the hypothesis that their phenotype should reflect inhibitory effects of tenapanor on intestinal Pi uptake. Mice were challenged by high dietary Pi, and hormones as well as plasma Pi were determined. The in vivo effects of tenapanor were studied via 33P uptake from the intestine. Pi transport was measured ex vivo in everted gut sacs (EGS) and brush border membrane vesicles (BBMV). Intestinal and renal protein expression of Pi transporters were determined. No differences were found between genotypes in terms of Pi homeostasis before Tam. Two weeks after Tam, NHE3IEC‐KO mice had similar Pi homeostasis, but a ~25% reduction in FGF23 compared with control mice on standard diet. Two‐fold greater intestinal Npt2b expression in NHE3IEC‐KO mice (P<0.05) was associated with significantly enhanced Pi uptake in EGS and BBMV. One hour after acute oral Pi loading (0.5 mol/L NaH2PO4), plasma Pi levels increased to a significantly greater extent in NHE3IEC‐KO compared to control mice (2.0±0.2 vs 1.1±0.1 mmol L‐1, P<0.05). Tenapanor treatment caused ~67% lower 33P uptake (P<0.05) after 5 minutes compared with vehicle‐treated control mice. At later time points, tenapanor‐treated control mice showed an increase in intestinal Pi uptake, reaching a maximum of 33P in plasma after 60 minutes. In response to high dietary Pi, plasma Pi slightly but significantly decreased (‐0.25±0.1 mmol/L, P<0.05) in control mice, possibly the consequence of the combination of significantly elevated PTH and FGF23. In contrast to control mice, high dietary Pi significantly increased plasma Pi in NHE3IEC‐KO mice (0.5±0.1 mmol/L, P<0.05), which was associated with significantly increased plasma PTH levels (P<0.05). FGF23 levels also increased to a significantly greater extent in NHE3IEC‐KO vs control mice (552±66 vs 244±14 pg/mL, P<0.05). This was associated with ~2.25‐fold greater Npt2b expression (P<0.05). Fifty percent lower renal Npt2c expression (P<0.05) and a trend for lower Npt2a expression (~25%) were unable to correct for higher plasma Pi. In summary, intestinal NHE3 significantly contributes to Pi homeostasis. In contrast to effects described for tenapanor on Pi homeostasis, NHE3IEC‐KO mice show higher, rather than lower, intestinal Pi uptake.
Glutamate transporter activator LDN‐212320 attenuates CFA‐induced cognitive deficits and anxiety‐like behavior in mice
Authors: Rahman Shafiqur , Alotaibi Ghallab
Published: May 2022

The astroglial glutamate transporter‐1 (GLT‐1) in the hippocampus and anterior cingulate cortex (ACC) is critically involved in acute and chronic nociceptive pain. We have previously shown that 3‐[[(2‐Methylphenyl) methyl] thio]‐6‐(2‐pyridinyl)‐pyridazine (LDN‐212320), a GLT‐1 activator, in the hippocampus attenuates acute and chronic nociceptive pain. The cellular and molecular mechanisms of GLT‐1 modulation in the hippocampus and ACC in chronic pain‐induced cognitive deficits and anxiety‐like behaviors are unknown. Here, we have examined the effects of LDN‐212320 in complete Freund’s adjuvant (CFA)‐induced cognitive deficits and anxiety‐like behaviors in mice. Furthermore, we have measured CFA‐induced impaired spatial, working, and recognition memory using Y‐maze and object‐place recognition test. In addition, we have determined chronic pain‐induced anxiety‐like behaviors using elevated plus maze and marble burying test. We have also evaluated the effects of LDN‐212320 on cAMP response element‐binding protein (pCREB) expression in the hippocampus and ACC during CFA‐induced cognitive deficits and anxiety‐like behaviors using Western blot analysis and immunofluorescence assay. Pretreatment of LDN‐212320 (20 mg/kg) significantly attenuated CFA‐induced impaired spatial, working, and recognition memory. The LDN‐212320 (20 mg/kg) significantly reduced CFA‐induced anxiety‐like behaviors. Additionally, LDN‐212320 (20 mg/kg) significantly reversed CFA‐induced decreased‐pCREB expression in the hippocampus and ACC. Overall, these results suggest that the LDN‐212320 prevents CFA‐induced impaired cognitive behaviors and neuronal deficits via GLT‐1 modulation in the hippocampus and ACC. Therefore, LDN‐212320 may have therapeutic utility for the prevention and treatment of chronic pain‐associated with cognitive impairments and anxiety‐like behaviors.
The ion binding site in transmembrane domain 5 of NBCe1 has negligible role in electrogenicity
Authors: Choi Inyeong , Lee Soojung
Published: May 2022

Background

The sodium bicarbonate transporters move HCO3– across cell membranes and regulate intracellular pH or transepithelial HCO3– transport. Some members of these transporters, such as NBCe1 and NBCe2, mediate electrogenic transport (1 Na+ and 1 CO32– or 2 HCO3–), while others, such as NBCn1 and NBCn2, mediate electroneutral transport (1 Na and 1 HCO3–). A structure/function study has shown that the loop between transmembrane (TM) 7 and 8 is responsible for electrogenicity. On the other hand, a recent report on the molecular dynamics simulations of NBCe1 cryoEM structure has proposed an ion binding site, which is located in transmembrane domain (TM) 5 and serves as a transient binding site to attract anions from the surrounding solution. In the present study, we tested whether this binding site is responsible for electrogenic Na/HCO3 transport.

Methods

Amino acid residues constituting the ion binding site of NBCe1 and NBCn1 were mutated and their effects on electrogenicity were evaluated by expressing the corresponding mutant transporters in Xenopus oocytes and assessing an Na/HCO3 transport current (INBC) in two‐electrode voltage clamp. In addition, the loop between TM7 and TM8 was replaced and its effect on INBC was examined.

Results

1) Replacing TM5 of NBCe1 with the corresponding TM5 of NBCn1 retained INBC, whereas a reciprocal replacement did not. 2) Replacing charged residues constituting the ion binding site in NBCn1 with the residues at the corresponding site in NBCe1 did not induce INBC. 3) Replacing the loop between TM7 and TM8 in NBCe1 with the corresponding loop in NBCn1 abolished INBC. 4) Replacing the loop in the NBCe1/TM5 chimeric transporter with the loop in NBCn1 abolished INBC.

Conclusion

We conclude that the NBCe1 electrogenicity is not associated with the ion binding site but determined by the loop between TM7 and TM8.
Potential Impact of Phosphorylation on Malate Dehydrogenase and Citrate Synthase Protein‐Protein Interaction
Authors: Blatt Alexander , Provost Joseph
Published: May 2022

Fine tuning of metabolic protein pathways is an insightful way to learn about the progression of diseases linked to metabolism, such as Alzheimer’s disease. Analyzing specific protein interactions in a structure‐function relationship, opens the discussion for potential therapeutic interventions. Malate Dehydrogenase (MDH) and Citrate Synthase (CS) interact in human mitochondria forming a metabolon found within the Krebs Cycle. While the interaction between MDH and CS is not novel, the complete interface between MDH and CS is not yet determined. Furthermore, how these interactions may be modulated by post‐translational modification has yet to be investigated. This study attempts to investigate if key residues driving isoform specific interactions between the MDH and CS are regulated by phosphorylation. Published docking models of MDH and CS highlight several potential sites of interaction, but the key residues of MDH in the interface are not experimentally determined. We performed additional docking predictions using Hawkdoc and SwissDock with published cross‐linked lysine residues of MDH‐CS. Interaction between cytosolic MDH and CS is reported to be much weaker, thus a primary structure alignment highlights the areas of unique residues that might be critical for mitochondrial MDH‐CS interactions. Meta‐analysis of phosphorylation databases from mammalian cells/tissues have identified several sites of phosphorylation with unknown function. To examine the key sites of interaction between MDH and CS and its regulation, we selected several putative phosphorylation sites specific to mitochondrial MDH; S45, T85, S222, and T224, and generated phosphomimic S/T to D mutants. Utilizing his‐tagged recombinant human MDH and CS, we measured interaction using fast thermal melt assays with the presence and absence of crowding agents (10% glycerol; 20% PEG) to determine regulation of phosphomutant interactions. Lastly, we conducted pull‐down assays to illustrate the possible changes in interactions with CS in the wild‐type and phosphomimic MDH‐CS pairs. In determining the interface and post‐translational modification between MDH and CS with these key residues, we can clarify where and how the governing mechanism regulates MDH and CS interaction. This will enable us to gain insight into the modulation of the binding interface interaction and contribution towards altering various metabolic disease pathways.
Protease‐Induced Excitation of Dorsal Root Ganglion Neurons in Response to Acute Perturbation of the Gut Microbiota is Associated with Visceral Hypersensitivity
Authors: Baker Corey C. , Sessenwein Jessica , Omar Amal A. , Tsang Quentin , Yu Yang , Segal Julia , Ghasemlou Nader , Sheth Prameet , Vanner Stephen , Reed David , Lomax Alan
Published: May 2022

Abdominal pain is a major symptom of diseases associated with microbial dysbiosis. Disruption of the gut microbiota with antibiotics increases visceral pain, and germ‐free mice are more prone to pain than conventionally‐raised mice. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons. Patch clamp electrophysiological recordings of dorsal root ganglion (DRG) neuron excitability were obtained from control mice and mice treated with the non‐absorbable antibiotic vancomycin (50 µg/ml in drinking water) for one week. Ten days prior to recording visceromotor response (VMR) telemetric transmitters were placed into the abdominal cavity of the mice and allowed to recover. VMR was measured by insertion of balloon catheter into the rectum under light anesthetization in both control and vancomycin treated mice, then distended to 80 mmHg and VMR recorded. Bacterial dysbiosis was verified by metagenomic analysis of stool microbial composition. Mice treated with vancomycin were more sensitive to colorectal distension in vivo (VMR increased by 70% at 80 mmHg compared to control), and DRG neurons from vancomycin‐treated mice were hyperexcitable in vitro compared to water‐treated controls (rheobase decreased by 30% relative to control). Interestingly, hyperexcitability of DRG neurons was not restricted to gut projecting neurons, suggesting a widespread effect of gut dysbiosis on pain pathways. Incubation of DRG neurons from naïve mice in serum from vancomycin‐treated mice increased neuron excitability (rheobase decreased by 30% relative to control), suggesting that microbial dysbiosis alters circulating mediators that influence nociception. Multiplex ELISA measurements did not detect any significant changes in serum cytokines or chemokines between vancomycin‐treated and control mice. The cysteine protease inhibitor E64 (30 nM) and the protease‐activated receptor 2 (PAR2) antagonist GB‐83 (10 µM) each blocked the increase in DRG neuron excitability in response to serum from vancomycin‐treated mice. Naïve DRG neurons incubated with fecal supernatants from vancomycin‐treated mice also exhibited increased excitability (rheobase decreased by 40% relative to control), but supernatants derived from colonic tissue failed to cause hyperexcitability. Overall, this data suggests that microbial dysbiosis within the gut alters pain sensitivity. This effect is not caused by inflammation or host derived factors, rather bacterially‐derived cysteine proteases activating PAR2 on DRG neurons.
Evaluation of extracellular vesicle isolation and detection repeatability from cell cultures, biofluids, and mammalian tissues
Authors: Aksamitiene Edita , Chaney Eric J. , Marjanovic Marina , Yin Leilei , Spillman Darold R. , Boppart Stephen A.
Published: May 2022

Unaccounted variations in biological marker sample handling, preparation, and data collection can result in inaccurate or unreliable data sets. Herein, we assess the repeatability (test‐retest reliability) of extracellular vesicle (EV) size and concentration measurements by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) methods. We evaluate the contribution of pre‐analytical variation and analytical imprecision factors toward the total experimental variability in routine preparation and quantitative analysis of crude large (microvesicle, MV) or small (exosome, EXO) EV particle‐enriched fractions following differential velocity centrifugation. This is performed on EV‐enriched cell culture media, human biofluids, and mammalian tissues, which represent an increasing degree of analyte complexity and heterogeneity. Incremental increase in NTA camera level (CL) setting (shutter and gain) was associated with a proportional detection of larger small‐sized particle counts. There was a strong linear relationship between the raw sample volume or wet tissue weight input, and EV output. At optimal non‐oversaturating particle detection conditions, NTA and DLS techniques delivered low (< 10%) inter‐sample coefficient of variability (CV%) when estimating EV size or number in technical replicates (TRs) prepared by using a 2‐step analyte dilution and sampling strategy. However, according to Bland‐Altman analysis, the repeatability of intra‐sample EV concentration measurements, but not size measurements by NTA, was prone to a significant run order bias within the first data capture cycle (DCC). Although analyte origin (random anatomical sub‐locations or different batches) did not represent a substantial source of error in EV analysis, a parallel EV isolation in TRs derived from a single supply, and a successive sample pooling with a potential preparation of second‐tier serially diluted TRs, is advised. To reduce the amount of analytical error in NTA analytes with the particle per frame (PPF) number between 40 and 100, and the polydispersity index (PDI) of < 30%, no less than three 60 sec‐long or six 30 sec‐long repeated intra‐sample measurements by at least two successive DCCs should be recorded. In DLS, ten 30 sec‐long repeated measurements should be captured in each of three independent runs.
Real‐Time dynamics of renal autoregulatory responses to BP changes in conscious, normotensive and hypertensive rat strains
Authors: Griffin Karen A. , Polichnowski Aaron J. , Williamson Geoffrey A. , Bidani Anil K.
Published: May 2022

Renal autoregulatory (AR) mechanisms are believed to insulate RBF and GFR from the continuously fluctuating systemic pressures and protect the glomerular capillaries from barotrauma by preventing glomerular transmission of systemic hypertension. However, renal AR has usually only been assessed by the degree to which RBF is restored to baseline after step changes in BP are imposed, generally under anesthesia. AR indices (% changes in RBF / % changes in BP) are used to quantitate the magnitude of the AR compensation. However, such methods do not provide an assessment of the real time dynamics of AR responses to the spontaneously occurring BP fluctuations in real time. We have recently described novel analytic methods to characterize both the magnitude and the kinetics of such AR responses to BP fluctuations in conscious Sprague‐Dawley rats with intact (controls) and impaired AR (3/4 nephrectomy) and/or calcium channel blockade (JASN 31(2):324‐336, 2020). Essentially, AR indices were calculated for adjacent segments of varying lengths (0.5 – 20 sec) which exhibited a MAP difference of at least 5 mmHg. These studies indicated a much greater rapidity of the normal AR response than observed using standard methods under anesthesia with the bulk of AR compensation (>50%) occurring within 2.5 sec with complete compensation occurring more slowly by ~10 sec. This rapid initial and likely myogenic component of the response was noted to be significantly slower in states of impaired renal AR with <40% of AR compensation being achieved by 5 sec. To assess the generalizability of these pattern of AR responses and interpretations of rapid myogenic protection, similar AR analysis were performed on concurrent BP and RBF recordings of 1‐4 hr duration that had been obtained in conscious chronically instrumented hypertensive and normotensive rat strains in our laboratory for transfer function analysis in control SHR, SHRsp, Dahl S (DS) and R (DR) rats, and Sprague‐Dawley (SD) rats over the past 20 years (Fig. 1). The pattern of AR responses and compensation is remarkably similar in all rat strains except that the responses are even faster and stronger in hypertensive rats with ~80% compensation being achieved by 2.5 sec (Fig. 2). To further examine the impact of ambient BP on AR responses, AI were separately calculated when the ambient MAP was within 30 mmHg below (low BP) or above (high BP) the average MAP during the recordings for each strain. There was a clear trend for the AR responses to be stronger at higher ambient MAP, although statistical significance was not achieved in all strains because of the scatter in individual animal data (Fig. 3). These data suggest that in addition to genetic factors, ambient BP per se may modulate the dynamics of protective renal AR responses in conscious rats.
A Knuckle‐Walking Signal in the Trabecular Architecture of the African Ape Scaphoid
Authors: Goldstein Deanna M. , Patel Biren A. , Sylvester Adam D.
Published: May 2022

African ape (Panand Gorilla) carpals have few diagnostic features in their external morphology reflecting knuckle‐walking hand postures, and whether the internal trabecular architecture of their carpals yields a functional signal for knuckle‐walking is unclear. Here we perform a high‐density, multiple‐VOI analysis of trabecular architecture in the scaphoid of knuckle‐walking African apes and non‐knuckle‐walkers (Pongo, Macaca,and Papio). We predict that (1) Gorilla, the taxon with largest body mass, will have the highest trabecular thickness (Tb.Th) and trabecular spacing (Tb.Sp) because Tb.Th and Tb.Sp have been shown to be correlated with body size, (2) bone volume fraction (BV/TV) will be higher in habitually terrestrial taxa than in arboreal taxa, as the forelimb is thought to experience relatively high compressive loads during terrestrial locomotion, (3) Pongo will have the lowest degree of anisotropy (DA) as they engage in variable locomotor behaviors, and (4) pattern of DA distribution will differ between knuckle‐walking and non‐knuckle‐walking taxa, as the consistency and direction of wrist joint loading likely differs among primates with different quadrupedal hand postures.

Fifty‐four scaphoids were scanned using high‐resolution µCT (voxel range: 48‐27 μm). Surface models were created from image data and then subjected to a weighted spherical harmonic analysis. Volumes of interest (VOIs) were manually placed into a surface model of the average scaphoid, and then warped to individual specimens using the thin plate spline interpolation function. Trabecular properties were calculated using the BoneJ plugin for ImageJ and MATLAB, and variation in distribution of trabecular parameters among taxa was summarized using a principal component analysis (PCA). Parameter values among species were compared with non‐parametric MANOVAs using a randomized residual permutation procedure, and PC scores were compared using Kruskal‐Wallis ANOVAs. The parameter distribution within each bone was visualized by comparing species‐average color maps of average parameter values.

As predicted, Tb.Th and Tb.Sp are greatest in Gorilla, indicating that these parameters are related in part to body size. In contrast, results indicate that BV/TV and DA magnitudes do not follow predictions. Specifically, habitually terrestrial taxa do not have significantly higher BV/TV than arboreal taxa, and taxa that engage in more variable locomotor behaviors (i.e., Pongo) do not have the lowest values of DA. Finally, results indicate that knuckle‐walking African apes can be distinguished from other taxa studied here by pattern of DA distribution, and these differences appear to reflect differences in habitual hand postures and locomotor modes. If this pattern is unique to knuckle‐walking apes among hominoids, and is also identified in the scaphoid of fossil hominoids, then it may be used to indicate whether fossil taxa were habitual knuckle‐walkers.
The role of NMDA receptors subunits in the progression of inflammatory breast cancer (IBC)
Authors: Mendez‐Santacruz Laura L. , Bittman‐Soto Xavier S. , Rodriguez‐Martir Keishla , Peterson‐Peguero Esther A. , Maldonado‐Vlaar Carmen
Published: May 2022

Inflammatory Breast Cancer (IBC) has a 73% incidence of metastasis to the brain as compared to other cancer types like breast, lung, and bone cancer. Previous research showed that tumor cells from different breast cancer subtypes (‐HR / ‐HER2) and (‐HR / + HER2) have a high incidence of crossing the blood‐brain barrier and inducing increased expression of glutamate receptors NMDA subunits (NMDAR1 and NMDR2) in the brain. IBC is the most aggressive and rare type of breast cancer. The absence of a solid tumor is replaced by swelling, redness, and skin changes, resulting in many cases in misdiagnosis of an infection in the invasion and migration phase; this type of cancer blocks blood and lymphatic vessels in the breast's skin, causing local inflammation and rapid metastasis. However, little is known of the role that these glutamatergic receptors have in the development and/or progression of IBC. The present work provides a new understanding of the cellular and molecular effects NMDA receptors (NMDAR) have on oncogenic phenotypes in IBC. The detection and quantification of the NMDAR were evaluated by RT‐qPCR, Western blot, and immunofluorescence (IF) techniques in deferments breast cancer cell lines (MCF7, MDA‐MB‐231) and IBC cell lines (SUM149, and SUM190). Also, NMDAR antagonists (memantine and dizocilpine “MK‐801”) were used in 2D and 3D cell cultures to see the pro‐oncogenic effects of migration and proliferation in cell models. In addition to this, the autocrine regulation of Glutamate in IBC cell lines is verified. Results showed a significant increase in the expression of NMDARs in IBC cell lines. In the functional cell culture assays with NMDAR antagonists, a decrease in migration and proliferation in IBC cell lines was found. These findings may be related to the high aggressiveness and the high rate of metastasis by IBC within the brain. Our results have high clinical relevance since having extensive knowledge of the role of NMDARs in IBC progression could complement therapeutic approaches for patients with IBC.
Telocytes: Newly Discovered Interstitial Cells in the Human Female Internal Reproductive Organs – Identification of a Suitable Immunohistochemical Marker and Possible Significance in the Pathogenesis of Uterine Fibroids
Authors: Klein Martin , Csöbönyeiová Mária , Visnyaiová Kristína , Danišovič Ľuboš , Varga Ivan
Published: May 2022

Introduction

Telocytes are a cell population discovered in 2005. Within the organs of the female reproductive system (FRS), telocytes were described as having an immunomodulatory role and also functioning as hormone sensors. From the pathological perspective, telocytes were described in the pathogenesis of a wide spectrum of different conditions, including a role in tumorigenesis. The gold standard of telocyte research is electron microscopy, but immunohistochemistry is also widely applied. The main objective was to identify the most suitable immunohistochemical marker and subsequently use it in the study of uterine specimens from patients with uterine fibroids (leiomyomas).

Material and

Methods

Uterine specimens were obtained from 26 patients who underwent hysterectomy for various indications, including ectopic pregnancy and uterine fibroids. From all the uteruses, representative specimens were obtained from the myometrium and endometrium of the uterine body, and also from the endo‐ and exocervical mucosa. 5 μm thick sections were further processed for immunohistochemistry. To identify the most suitable marker of telocytes, 8 primary antibodies were used: CD117/c‐kit, CD34, vimentin, α‐smooth muscle actin, progesterone and estrogen receptor, desmin and S100 protein (DAKO, Denmark).

Results

The most specific marker was c‐kit. C‐kit‐positive cells were spindle‐shaped, triangular and star‐shaped. C‐kit positive cells were located in the endometrium, myometrium, exocervical, and endocervical mucosa. Comparing the distribution within the individual parts of the uterus, the most numerous population was observed within the myometrium. A considerable number of telocytes was also observed within the endocervical mucosa, while the fewest telocytes were described in the endometrium and exocervical mucosa. In the specimens of uterine fibroids, the non‐affected parts of the myometrium contained c‐kit positive cells within all the myometrial sublayers. The average c‐kit positivity expressed in percentage was 2.19 ± 0.51%. Those parts of the myometrium affected by uterine fibroids displayed complete c‐kit negativity.

Conclusion

C‐kit is the most suitable marker in the research of telocytes in the FRS. The loss of telocytes in the myometrium may have a role in the pathogenesis of uterine fibroids via three mechanisms: 1) hormonal imbalance, 2) stem cell dysregulation and 3) diminished angiogenesis. Therefore, therapeutic targeting of telocytes can be a potential future approach in the treatment of this condition.
In utero exposure to dietary emulsifier alters the behavioral activity in offspring
Authors: Mishra Jayshree
Published: May 2022

Objective

Pediatric mental health disorder is a combination of neuroinflammatory conditions with unknown etiology and currently, there are less awareness on the impacts of food additives on pediatric neuroinflammatory conditions and adolescent mental health disorders. Food additives such as dietary emulsifier (DE) (polysorbate 80, carrageenan and maltodextrin) are added to a variety of processed foods, and their consumption has increased dramatically worldwide which could be a risk factor for pediatric mental health disorders. Therefore, there is a critical need to understand the impact of maternal exposure to food additives associated pediatric mental health and the underlying mechanism.

Materials and Methods

Behavioral testing such as tail flick assay, radial arm, rotarod and hot plate assay was done on the offspring from both control and DE exposed mice for the impaired motor activity, pain sensitivity and cognitive behavior. Colonic microbial composition was determined through 16sRNA pyrosequencing of fecal DNA samples from control and in utero DE‐exposed offspring. Level of pro inflammatory cytokines in the colon tissue as well as the serum sample was measured using Multianalyte cytokine assay kit from Qiagen.

Summary of Results

Our results show that in utero exposure to DE leads to decreased brain sizes and impaired motor learning in the offspring. Additionally, in utero exposure to DE leads to decreased pain sensitivity as a sign of stress in offspring. In utero exposure to DE shows on onset of cognitive impairment in offspring. In utero exposure to DE leads to an elevated M1 macrophage cytokine profile and CLGI symptoms in the gastrointestinal tract of offspring. In utero exposure to DE leads to gut dysbiosis in offspring. Among the DE‐exposed adolescents, the male offspring had significantly higher impact on these parameters compared to the DE‐exposed female offspring. In utero exposure to DE leads to imbalance in serum levels of dihydrotestosterone (DHT) and estradiol (E2) in the Offspring.

Conclusion

Our data suggest that maternal exposure to food additive is associated with the male biased cognitive impairment and neurobehavioral changes in the offspring. In future, we will determine the specific mechanism responsible for the sex specific alternation in offspring born to DE exposed mother to develop a preventative and therapeutic strategies.
Limb Hemodynamic Responses to a Single Bout of Inspiratory Muscle Strength Training
Authors: Plouffe Audrey A. , Vranish Jennifer R.
Published: May 2022

Background

Inspiratory muscle strength training (IMST) has been shown to bring about improvements in respiratory muscle strength and reductions in blood pressure after 6 weeks of daily high intensity training. However, there has been little investigation into the effects of IMST on limb blood flow, and it is unclear how hemodynamics are altered within a single bout of training.

Purpose

To investigate the acute effects of one session of either low or high intensity IMST on blood flow in the upper and lower limbs.

Methods

Participants (n=7, 57% female) aged 20 to 47 years completed two randomized laboratory visits wherein resting blood flow was measured via duplex Doppler ultrasound at the left brachial and popliteal arteries. Following baseline measures, participants' maximal inspiratory pressure (PImax) was measured using a PowerBreathe K3 device. Participants performed one session (30 breaths) of IMST at either 15% or 75% of their PImax, and blood flow was measured again at each artery for five breaths during the final ten breaths of the training. At their second visit, subjects performed whichever training level had yet to be completed and blood flow was assessed as described above.

Results

Mean blood flow at the brachial artery appeared reduced during both 15% (p=0.070) and 75% training (p=0.078), which may be attributed to a significant increase in retrograde blood flow in both cases (p=0.014 and p=0.037, respectively). This increase in retrograde blood flow tended to be greater during the 75% training (p=0.066). Popliteal artery blood flow showed similar trends to brachial artery blood flow.

Conclusions

While IMST appears to reduce brachial artery mean blood flow during a single training session, this may be due to alterations in retrograde blood flow, which tended to increase in an intensity‐dependent manner. Further investigation into the chronic hemodynamic effects of IMST is warranted.
Myonuclei Can Replicate DNA
Authors: Borowik Agnieszka K. , Peelor Frederick F. , Dupont‐Versteegden Esther E. , McCarthy John J. , Miller Benjamin F.
Published: May 2022

Skeletal muscle is a heterogenous tissue composed of multinucleated muscle fibers and accompanying supporting cells like satellite cells (SCs). Myonuclei are believed to be post‐mitotic, arrested in G0 phase of the cell cycle and thus unable to proliferate. Consequently, it is hypothesized that growth of muscle is dependent on the donation of nuclei from SCs. Although some studies suggest that under certain conditions myonuclei can replicate, it has been difficult to address whether this replicative potential exists in vivo. We hypothesized that skeletal muscle resident myonuclei possess the ability to replicate in vivo. To test this hypothesis, we used the HSArtTA;Tet‐O‐H2B‐GFP (HSA‐H2B) mouse model, which uses doxycycline (DOX) treatment for temporal labeling (Tet‐ON) of residential myonuclei. After removing DOX, and thus turning off GFP‐labeling, we provided the stable isotope deuterium oxide (D2O) for 8 weeks to measure DNA synthesis. At the end of D2O labeling, muscles were harvested and GFP‐positive myonuclei were sorted using an antibody‐independent approach and a restrictive gating strategy. Both GFP+ (myonuclei) and GFP‐ (non‐myonuclei) fractions were then analyzed for D2O incorporation into DNA by GC‐QQQ. By our approach, DNA from GFP‐positive myonuclei was deuterium‐enriched and therefore deemed to be replicating. DNA replication occurred in at least one muscle of the 7 mice studied. We observed deuterium enrichment into DNA in 6 out of 7 plantaris (PLA), 4 out of 7 extensor digitorum longus (EDL), 4 out of 7 tibialis anterior (TA), 6 out of 7 gastrocnemius (GAS) and 7 out of 7 quadriceps (QUAD). The average fractional synthesis rates (FSR, %/day) of replicating myonuclei were: 0.0351 ± 0.017 in PLA, 0.0156 ± 0.015 in EDL, 0.0220 ± 0.018 in TA, 0.0223 ± 0.018 in GAS, and 0.02692 ± 0.043 in QUAD. These data are the first to unambiguously demonstrate that myonuclei replicate DNA in vivo. The proposed project challenges current dogma and might significantly impact skeletal muscle treatment to maintain muscle mass with age and disease.
A cadaveric study on the origins of the uterine artery
Authors: Rubalcava Ivan P. , Sakaguchi Alan Y. , Kar Rekha
Published: May 2022

The uterine artery (UA) is a visceral branch from the anterior division of the internal iliac artery (IIA). Knowledge of the variant origins of this artery is critical during radiologic and surgical procedures of the pelvis, especially those involving the uterus, where managing blood flow is vital. Though its path and destination are well agreed upon, the origin of the UA differs notably, and has led many anatomists to create numerous descriptions for the distinctive variation. This project investigated the origin of the UA in the unique South Texas population of female cadavers donated to the UT Health San Antonio and established a fundamental and comprehensive classification system of the origin of this vessel which readily accommodates previous systems while improving its descriptive terminology. As the UA was identified, its origin was classified into three different ranks—primary, secondary, or tertiary. Primary‐ranked vessels originated directly from the internal iliac artery (IIA) and took three different formations as a separate branch, bifurcation with another vessel, or trifurcation with two other vessels. Secondary‐ranked UAs originated from primary vessels and tertiary‐ranked vessels originated from secondary vessels. 58.97% (23/39) of the UAs studied arose from the IIA as a primary‐ranked vessel, 38.5% (15/39) as a secondary rank, and 2.6% (1/39) as a tertiary rank. 21.7% (5/23) of primary‐ranked UAs took origin from IIA as a separate branch, 56.5% (13/23) as bifurcation with another vessel and 21.7% (5/23) as trifurcation with two other vessels. The UA originated as a bifurcation with the umbilical artery (UmbA) in 13/13 cases. UA origin as a trifurcation was observed in 3 different patterns: 40% (2/5) with the UmbA and the superior vesical artery (SVA); 40% (2/5) with the UmbA and vaginal artery (VA); 20% (1/5) with SVA and the internal pudendal artery (IPA). 93.3% (14/15) of secondary‐ranked UA arose from the UmbA and 6.7% (1/15) from the IPA. We observed a single tertiary ranked UA arising from the vaginal artery (VA), which took origin from the UmbA, a branch of the anterior division of the IIA. It is imperative that the prevalence of these primary‐ranked UA bifurcations and trifurcations be further established since permanent occlusion at the vessel’s origin may subsequently lead to necrosis of unintended tissues. Larger sample sizes amongst differing populations may lead to the discovery of uncommon bifurcation and trifurcation patterns, or with similar vessels in differing proportions. It would also be important to translate the evaluation criteria used in this study into 3D and 4D digital subtraction angiography or cone‐beam computed tomography software for clinical studies. Increasing awareness of the variant origins of the UA will ultimately improve patient treatment outcomes, particularly for female reproductive health.
Dynamic Desensitization of Gaq Signaling and Gaq‐dependent GPCR Crosstalk by GRKs
Authors: Xiang Guoqing , Acosta‐Ruiz Amanda , Moon Jared D. , Kristt Mindy , Lee Francis , Levitz Joshua
Published: May 2022

G protein‐coupled receptor (GPCR) signaling is subject to many regulatory mechanisms that occur over a range of time scales. Compared to analysis of slow, trafficking‐based mechanisms relatively little attention has been paid to rapid mechanisms that take place on the seconds time scale. In our study, we utilized live cell calcium imaging to investigate the acute effects of a subfamily of GRKs, GRK2 and 3 on both Gq signaling and Gi/s‐Gq crosstalk. We first focused on group 1 metabotropic glutamate receptors (mGluRs) and found that overexpression of GRK2 and 3 drives an acute desensitization of Gq‐dependent signaling. This desensitization effect occurs on the seconds time scale, is rapidly reversible, and depends on direct interaction between GRK and Gq but is independent of its kinase activity. We find that this mode of rapid desensitization is generalizable across Gq‐coupled GPCRs and is insensitive to the widely used GRK2/3 kinase inhibitor CMPD101. Finally, we studied whether the extent and timing of crosstalk between Gi and Gs‐coupled receptors with Gq‐coupled receptors is also regulated by GRK2. We find that GRK2 dramatically reduces the level of functional crosstalk and accelerated the timing of desensitization of calcium responses to Gi or Gs‐coupled receptor activation. This regulatory effect of GRK on crosstalk is dependent on its interaction with both Gq and G beta gamma, but is independent of its kinase activity as well. Together this work reveals potent forms of GPCR signaling regulation with major implications across physiological contexts.
High‐fat diets increase nicotine self‐administration in male and female mice by impacting dopamine release in the nucleus accumbens core
Authors: Henderson Brandon J. , Cooper Skylar Y. , Richardson Montana R.
Published: May 2022

Prior investigations focused on the impact of high‐fat diet (HFD) on nicotine reward and reinforcement have indicated that HFD exposure increases reward‐related and reinforcement‐related behaviors associated with nicotine. However, there has been no investigation into how nicotine‐related behaviors may be altered by HFD. To investigate the potential mechanisms of HFD on nicotine addiction‐related behaviors, we exposed adult male and female mice to HFD or a regular diet (RD) for 6 weeks. Following this, we trained mice in a nicotine e‐vape® self‐administration (EVSA) paradigm. Here mice, were trained to self‐administer 6 mg/mL nicotine on a fixed‐ratio 1 (FR1), FR3 and progressive‐ratio schedules. Following behavioral assays we anesthetized the mice and extracted brains for ex‐vivo fast‐scan cyclic voltammetry (FSCV) assays. We observed that both male and female mice exhibited an increase in nicotine intake on the FR1 schedule but had the same intake of nicotine as RD mice on a FR3 schedule. We also observed that both male and female mice exhibited a significantly lower breakpoint when assessed on a progressive ratio. Using FSCV, we observed that HFD mice exhibited a significant decrease in dopamine release in the nucleus accumbens core when compared to RD mice at both tonic and phasic stimulation frequencies. These data support the fact that HFD promotes nicotine intake at low‐effort schedules (FR1) and this may be due to decreased baseline dopamine release in the nucleus accumbens core.
Role of inflammation and ER stress in HFpEF pathogenesis
Authors: Srinivas Balaji , O'Regan Jacob D. , Belmadani Souad , Matrougui Khalid
Published: May 2022

Background

Heart failure with preserved ejection fraction (HFpEF), characterized by diastolic dysfunction, inflammation, cardiac hypertrophy and fibrosis, myocardial capillary rarefaction, pulmonary hypertension, aortic stiffness, and impaired endothelial function, is the major unmet need in cardiovascular medicine and remains an untreatable cardiovascular disease. The role of inflammation and endoplasmic reticulum (ER) stress in HFpEF pathogenesis is not well understood.

Methods

C57/Bl6 mice were divided into five groups. Group 1: control mice. Group 2: Mice fed high fat diet (HFD) and received L‐NAME (eNOS inhibitor,0.5 g/L) in the drinking water for 4 months. Group 3: Mice fed HFD + L‐NAME for 4 months then treated with ER stress inhibitor for one month (Tudca, 150 mg/Kg). Group 4: Mice fed HFD + L‐NAME then treated with anakinra for one month (IL‐1beta receptor antagonist, 1 µg/mouse/every other day). Group 5: Mice fed HFD + L‐NAME then treated with Tudca + anakinra for one month. We measured body weight, blood pressure, cardiac function, diabetes status, and vascular endothelial function.

Results

Mice in group 2 fed HFD and L‐NAME in the drinking water for 4 months developed metabolic syndrome, hypertension, cardiac hypertrophy, lung edema, low running performance, vascular endothelial dysfunction, cardiac diastolic dysfunction, but no change in cardiac ejection fraction (EF) compared to group 1. Interestingly, the treatment of mice in groups 3, 4, and 5, with Tudca with and without anakinra provides protection from hypertension, body weight increase, metabolic syndrome, cardiac hypertrophy, lung edema, and vascular endothelial and diastolic dysfunction induced by HFD and L‐NAME.

Conclusion

Our data illustrate that ER stress and inflammation play a critical role in HFpEF pathogenesis.
Activation by Cleavage of the Epithelial Na+ Channel α and γ Subunits Independently Coevolved with the Vertebrate Terrestrial Migration
Authors: Kashlan Ossama , Wang Xue‐Ping , Balchak Deidra M. , Gentilcore Clayton , Clark Nathan L.
Published: May 2022

Vertebrates evolved mechanisms for sodium conservation and gas exchange in conjunction with migration from aquatic to terrestrial habitats. Epithelial Na+ channel (ENaC) function is critical to systems responsible for extracellular fluid homeostasis and gas exchange. ENaC is activated by cleavage at multiple specific extracellular polybasic sites, releasing inhibitory tracts from the channel’s α and γ subunits. Here we investigated the evolution of ENaC regulatory mechanisms to determine which features coevolved with the marine‐terrestrial transition. We consistently found both activating cleavage sites in the ENaC α and γ subunits of terrestrial vertebrates, while they appeared only sporadically in fishes. We confirmed that cleavage occurred at sites found in the γ subunit from Australian lungfish, leading to channel activation. Phylogenetic analysis and likelihood ratio tests showed that proximal and distal polybasic tracts in ENaC subunits coevolved, consistent with the dual cleavage requirement for activation. They also showed a coevolutionary dependence of tandem polybasic tracts with terrestrial status and with lungs, coincident with the ENaC activator aldosterone. Amplification of transcripts by RT‐PCR in ray‐finned Polypteriformes and previously in lobe‐finned lungfish suggest that transcripts for ENaC subunits with cleavage sites are readily detected at important sites of ion exchange (gills and kidney), but are difficult to detect in lungs. Analysis of ancestral reconstructions strongly suggests that the polybasic tracts appeared independently in the α and γ subunits of ENaC. Similar analyses of the PY motif, required for Nedd4‐2 dependent regulation, showed no coevolutionary pattern and that the PY motif first arose in an ancient ancestral ENaC subunit. Our data suggest that changes associated with adaptation to terrestrial life provided selective pressure for the development of ENaC activation by proteolytic cleavage.