bluuebunny/crossref_metadata_2025 huggingface dataset:
Daily E‐cigarette Vapor Exposure Does Not Modify Response to 10% Normobaric Hypoxia in Long‐Evans Rats
Authors: Stokes Jennifer A. , Peters Alicia
Published: May 2022

E‐cigarettes or electronic nicotine delivery systems are devices used to deliver aerosolized liquids often containing nicotine and other chemicals. The short and long term effects of e‐cigarette vapor exposure are still under active investigation with many studies focusing on changes in cardiovascular function, lung tissue morphology, and inflammatory markers. This pilot study aimed to investigate the effects of seven days of e‐cigarette vapor exposure in adult rats on lung function and the physiological response to acute hypoxia. Since the chemicals found in e‐cigarette solutions have been shown to alter properties of the lung tissue, we predicted that response to acute hypoxia may be altered with vape exposure. Using random assignment, ten adult male Long‐Evans rats were assigned to vape (experimental) or air (control) groups. The animals were exposed to either air (n=4) or JUUL 5% nicotine vapor (n=6) using a whole‐body exposure chamber, twice a day for ten minutes for seven consecutive days. Ventilation was recorded on day 0 (baseline, pre‐exposure) and day 8 (post‐exposure) using unrestrained whole‐body plethysmography. Minute ventilation, tidal volume, and breathing frequency were assessed in normoxia and normobaric hypoxia (10% oxygen). Blood was collected on day 8 for cotinine (a nicotine metabolite) detection in serum. Cotinine was found to be present in the serum samples of the vape groups (86.6 ng/ml ± 1.0 ng/mL) but not the air groups (0.0 ng/mL) confirming e‐cigarette vapor exposure in the experimental group only. Baseline ventilation data collected on day 0 and post‐exposure ventilation data collected on day 8 were compared between air and vape groups across three different parameters: minute ventilation, frequency, and tidal volume. These parameters were first compared in normoxic conditions resulting in three distinct two‐way ANOVAs comparing the variables time and treatment. These parameters were also compared in hypoxic conditions resulting in an additional three distinct two‐way ANOVAs comparing the variables time and treatment. No significant difference was found among any of the comparisons (p > 0.05 for all). In this pilot study, a seven day vape exposure in adult rats did not affect the response to acute hypoxia. We plan to repeat this study with modifications to the vape protocol and exposure duration.
3D cardiac calcium imaging of zebrafish revealed doxorubicin‐induced cardiotoxicity
Authors: Liau Ian
Published: May 2022

The homeostasis of calcium ions is important for regulating the electro‐mechanical coupling of the heart. Hence, disturbance of the calcium homeostasis may impair both cardiac electrophysiology and cardiac function. Here we present the use of light‐sheet imaging and image processing algorithm to reconstruct dynamic 3D cardiac images and determine the calcium dynamics of zebrafish heart. We quantified the stroke volume and the ejection fraction from reconstructed dynamic 3D cardiac images. Furthermore, we restored motion‐artifact‐free local calcium transients of a beating heart, from which the propagation of the cardiac calcium signaling was determined. Lastly, we studied chemotherapy‐induced cardiotoxicity and showed that doxorubicin caused multiple cardiotoxic effects on larval zebrafish, including impaired cardiac chamber structure, decreased cardiac function, prolonged local calcium transients, and delayed calcium signaling propagation. We anticipate that our novel approach will spark applications that require accurate and simultaneous assessment of cardiac function and calcium dynamics.
Current Status of Graduate Anatomy Education in The State of Ohio
Authors: Hall Tyler R. , Balta Joy Y.
Published: May 2022

It is unclear as to what is included in a graduate anatomy education in the United States. The purpose of this study was to examine the current status of graduate anatomy programs in the state of Ohio to determine their similarities and differences. A survey regarding graduate anatomy education was sent to a graduate anatomy faculty member at all Ohio institutions with a graduate anatomy program. These institutions included: Case Western University, Wright State University, Northeast Ohio Medical University, and The Ohio State University. Four institutions (100%) responded to the survey. One institution (25%) reported having an anatomy doctoral program, four (100%) institutions reported having an anatomy master’s program, and one (25%) institution reported having an anatomy graduate certificate program. The mean (standard deviation) number of students enrolled annually in anatomy master’s programs was 20.8 (7.6), and, on average, those students are required to complete 31.3 (2.5) semester credit hours in order to complete their anatomy master’s program. Three (75%) programs reported that students in their master’s anatomy programs are required to complete coursework in gross anatomy, histology, embryology, and neuroanatomy, while one (25%) institution only required coursework in gross anatomy and neuroanatomy. Four (100%) institutions reported that the students in their anatomy master’s program complete a full body dissection on a human body donor as part of their gross anatomy training. It appears that there are some consistencies regarding graduate anatomy programs, though more information is needed regarding graduate anatomy programs outside the state of Ohio. The Institutional Review Board (IRB) at The Ohio State University determined that this study did not require IRB approval or an IRB exemption.
Profiling Metabolic and Signaling Phenotype of Bladder Cancer Cell Lines
Authors: Ducci Giacomo , Pasquale Valentina , Rota Stefano , Arrigoni Edoardo , Campioni Gloria , Ventura Elisa , Giordano Antonio , Morrione Andrea , Sacco Elena , Vanoni Marco
Published: May 2022

Bladder cancer (BC) is one of the most common malignancies worldwide[1]. Most patients are diagnosed with non‐muscle invasive BC, which is associated with frequent recurrence. The prognosis for early‐stage bladder tumors is generally good; however, patients often progress to invasive disease resulting in a much less favorable outcome[2,3]. 3D cultures constitute more clinically relevant models than monolayers for studying cancer as spheroids recapitulate in vivo structures, cell‐cell interactions, nutrients and oxygen gradients[4]. In this study we conducted a comparative analysis of metabolism and cellular features of a panel of BC cell lines covering different stages/grades, grown in monolayer cultures and spheroids. Samples were analysed at different timepoints for cellular features, like spheroid morphology and viability, by quantitative imaging using Operetta CLS high‐throughput microscope. Here we show that high‐grade cell lines had an increased capacity to form viable spheroids. In addition, we used Seahorse technology to assess bioenergetic parameters, e.g. mitochondrial respiration and glycolysis, and demonstrated that high‐grade cell lines grown in monolayer cultures present higher basal and maximal glycolysis compared to low‐grade cells, while mitochondrial respiration showed no obvious correlation with pathological grade. No obvious correlation was observed in the use of either mitochondrial respiration or glycolysis and pathological grade during spheroid formation, although some high‐grade cell lines, like HT1376, showed a preferential use of respiration for ATP production. Our laboratories have previously established the role of the progranulin/EphA2 axis in regulating motility, invasion and in vivo tumour formation in bladder cancer[6] but whether this pathway regulates the capacity of bladder cancer cells to form spheroids has not been established. Significantly, we provide now preliminary evidence that genetic targeting of either progranulin or EphA2 by CRISPR/Cas9 approaches modulated spheroid formation of invasive bladder cancer cells. Understanding the pathways and mechanisms regulating spheroid formation and metabolic changes of bladder cancer cells might contribute to the identification of novel targets and specific therapies for bladder cancer.

1. C. Yeung, et al (2014), Pharmacoeconomics, 32, 1093–1104.

2. M. Babjuk, et al. (2017), Eur. Urol., 71, 447–461.

3. J. Alfred Witjes, T. Lebret, E. M. Compérat, N. C. Cowan, et al. (2017), Eur. Urol., 71, 462–475.

4. I. Vasyutin, L. Zerihun, C. Ivan, A. Atala. (2019), Anticancer Res., 39, 1105–1118.

5. V. Pasquale, G. Ducci, G. Campioni, A. Ventrici, et al. (2020), Cells, 9, 1–26.

6. S. Buraschi, T. Neill, S.‐Q. Xu, C. Palladino, et al. (2020), Matrix Biol., 93, 10–24.
Investigating the transcriptomic responses in rice roots during interactions with plant growth‐promoting bacteria, Burkholderia unamae
Authors: Mukherjee Arijit , Cook John , Yang Qinqing , Rahmatallah Yasir , Glazko Galina
Published: May 2022

Major crops such as rice and maize can benefit from associations with different plant growth‐promoting bacteria (PGPB). Studies have shown that these PGPB (e.g., Azospirillum, Burkholderia) promote plant growth primarily via nitrogen fixation and phytohormone secretion. However, our current understanding of the underlying molecular mechanisms involved in these associations is limited. For example, very little is known about the associations between plants and the symbiotic Burkholderia species, B. unamae,at a molecular level. Previously, we set up an experimental system where PGPB such as A. brasilense could colonize rice roots and promote plant growth. In this study, we used the same experimental system and showed that B. unamae could promote growth and colonize rice roots. Next, using RNA sequencing, we identified the transcriptomic responses in rice roots, one day post‐inoculation. We identified 1128 differentially expressed genes (DEGs) in rice roots and validated the expression of few genes via RT‐PCR. We identified genes involved in the flavonoid biosynthesis, defense, hormone signaling, and photosynthesis differentially expressed. We also identified several genes encoding for transcription factors, protein kinases, and transporters. Comparison of our dataset to existing RNA‐seq datasets in rice roots during interactions with other plant‐beneficial Burkholderia species (Burkholderia vietnamiensis, Paraburkholderia kururiensis, Paraburkholderia phytofirmans) indicated downregulation of defense genes during these interactions. Finally, we identified 398 DEGs in rice roots during interactions with B. unamae and A. brasilense. Some of these genes are likely to play important roles during these interactions. Overall, our study has identified several promising targets for future genetic studies.
Pilot Study on the Influence of Race and Sex on beat‐to‐beat Blood Pressure Variability in Healthy Young Adults
Authors: Jeong Soolim , Linder Braxton A. , Tharpe McKenna A. , Hutchison Zach J. , Barnett Alex M. , Culver Meral N. , Nichols Olivia I. , Fuller‐Rowell Thomas E. , Robinson Austin T.
Published: May 2022

BACKGROUND

Augmented beat‐to‐beat blood pressure (BP) variability is prognostic of target organ damage and cardiovascular events. A recent study demonstrated greater BP variability in healthy young Black males compared to White males, but whether these differences appear in females remains unclear. Therefore, we sought to examine race and sex differences in beat‐to‐beat BP variability in healthy young Black and White adults.

METHODS

52 adults including 13 Black females (age 21.5 ± 0.8 yrs, BMI 27.0 ± 4.2 kg/m2, resting BP 126 ± 10/78 ± 12 mmHg; Mean ± SD), 13 White females (21.2 ± 0.7 yrs, 24.0 ± 3.8 kg/m2, 116 ± 8/66 ± 11 mmHg), 9 Black males (21.6 ± 0.7 yrs, 25.5 ± 4.3 kg/m2, 131 ± 11/75 ± 11 mmHg), and 17 White males (21.5 ± 0.7 yrs, 25.0 ± 3.7 kg/m2, 123 ± 11/67 ± 9 mmHg) participated in this study. Non‐invasive beat‐to‐beat BP was continuously measured at the participant’s middle finger via photoplethysmography (Finapres NOVA) for 10 minutes while sitting semi‐recumbent in a dimly lit room. The finger pressure was calibrated using resting oscillometric brachial BP. Heart rate was measured via 3‐lead electrocardiogram (ECG) and BP was gated to R‐waves from the ECG signal. BP variability was assessed with the average real variability (ARV) index and standard deviation (SD) of all BP readings. Statistical procedures included two‐way analysis of variance (sex x race) with Bonferroni post‐hoc test when appropriate (a priori α < 0.05).

RESULTS

No significant differences existed for systolic BP ARV (sex p= 0.85, race p= 0.41, interaction p= 0.63; Black females 2.02 ± 0.64, White females 1.84 ± 0.44, Black males 1.98 ± 0.24, White males 1.93 ± 0.44) or diastolic BP ARV (sex p= 0.31, race p= 0.76, interaction p= 0.51). For systolic BP SD, there was a significant main effect of sex (p= 0.008) suggesting greater systolic BP variability in males than females, and a trend for race by sex interaction (p= 0.08) but no difference for race (p= 0.79); (Black females 4.80 ± 1.07, White females 5.80 ± 2.10, Black males 7.77 ± 3.89, White males 6.42 ± 1.74). However, there were no significant differences for diastolic BP SD (sex p= 0.19, race p= 0.55, interaction p= 0.46).

CONCLUSION

These preliminary findings suggest males have significantly greater systolic BP variability than females from systolic BP ARV. No significant differences were identified for diastolic BP SD and BP ARV (systolic and diastolic) by race and sex, but additional data are needed.
In vitroevaluation of anti‐aging and regenerative properties of dermatan sulfate for skin care
Authors: Galvez‐Martin Patricia , Martinez‐Puig Daniel , Soto‐Fernández Cristina , Romero‐Rueda Jessica
Published: May 2022

Introduction

Dermatan sulfate (DS) is a linear polysaccharide classified as a sulfated glycosaminoglycan (GAGs). GAGs form an integral part of the skin and make up 0.1–0.3% of total skin weight. DS is covalently attached to the core proteins to form proteoglycans (PGs). PGs predominates in dermis presenting higher level of DS than other sulfated GAGs.

The intrinsic aging processes of the skin entails the reduction of total sulfated GAGs levels causing a thinning of the dermis and modifying the quality of the skin. DS possess strong biological activity that can be modulated to improve skin quality.

This study was performed to determine the effects of DS on Human Dermal Fibroblasts (HDF) to evaluate its anti‐aging and regenerative properties.

Methods

Human Dermal Fibroblasts (HDF) were isolated from a sample of the foreskin of a neonatal caucasian donor (Lonza). The effect of the DS on cell proliferation was assessed at 24 h and 48 h in HDF, using the method bromodeoxyuridine (BrdU) incorporation, detected by ELISA. The cell migration was evaluated in HDF at 48 h using the Oris™ Cell Migration Assay (Platypus Technologies). To monitor the migration progress, the cells were labelled with calcein. The wound healing capacity of DS was evaluated in HDF using the scratch test. The inductive capacity on Extracellular matrix proteins (ECMp) synthesis was evaluated by immunolocalization‐ELISA quantifying type I and type III collagen and elastin.

Results

The results showed that DS induced HDF proliferation after 24 h and 48 h of treatment at all tested concentrations. A dose‐dependent increase in proliferation was observed after 48 h of treatment of these cells, reaching a maximum increase of 5.10 (± 0.82) fold at the highest concentration tested (5 mg/mL) compared to uHDF. Regarding HDF cell migration, no significant differences were observed at 24 h after treatment with DS.

The results showed a significant improvement in the in vitro wound healing capacity of HDF treated with 0.25 and 1 mg/mL of DS, with an increase in the healed area of 30.28% (± 4.27%) and 23.76% (± 8.87%) respectively, compared to uHDF.

Regarding the ECMp synthesis‐stimulating capacity of DS, the results showed a significant increase of both type III Collagen (50.98 ± 34.48% increase) and Elastin (32.25 ± 2.61%) levels in HDF treated for 72 h with 1 mg/mL of DS compared to uHDF. HDF treated with 0.25 mg/mL also showed higher type I Collagen production than uHDF, although the increase (30.96 ±7.04%) was not statistically significant.

Conclusions

The results showed that DS improves the proliferation and regeneration of HDF present in dermis. In addition, DS increases the synthesis of ECMp as collagen type III and elastin, promoting the maintenance and functionality of the dermis. All these properties support the regenerative and anti‐aging activity of DS acting against intrinsic aging in dermis.
Anatomical Substract of the Temporal Muscle Myofascial Trigger Points in Fresh Cadavers
Authors: Garrido Luis C. F. , Jacomo Alfredo L. , Saleh Samir O. , Hojaij Flávio , Andrade Mauro , Akamatsu Flávia E.
Published: May 2022

Background

Myofascial syndrome is a common cause of chronic musculoskeletal pain and is characterized by myofascial trigger points (MTPs). MTPs are clinically identified by palpation of a muscle or fascial taut band. Although pathophysiology of MTPs remains unclear, coincidence between them and muscle motor plate at the innervation zone (IZ) have been reported. The temporal muscle is related to headache and temporomandibular disorders and pain radiates from hyperalgesic points to distant areas symptomatic patients. Clinical reports usually describe four trigger points located in the temporal muscle and we aimed to identify anatomical correlation with muscle innervation.

Methods

We dissected the temporal muscles from five fresh cadavers to observe the exact point where nerve fibers penetrated the muscle belly. As muscle size varies among individuals, we calculated the relative entry point of the nerve into the muscle by defining six different areas in the muscle belly: three superior (I, II and III) and three inferior (IV, V and VI), beginning at the posterior margin of the muscle. Statistical analysis of anatomical data was obtained by Poisson distribution and logarithm link function followed by Bonferroni multiple comparisons. The comparison of points between the sides was made by Student’s t‐test. IBM‐SPSS for Windows version 20.0 software was used to perform the analysis, and Microsoft Excel 2003 software was used to tabulate the data. Data are presented as the mean ± standard deviation, and the level of significance was adjusted to 5% (p <0.05) for all tests.

Results

The number of temporal nerve branches did not differ between sides. Areas II and V had the largest number of temporal nerve entry sites, with a mean of 3 (22%) and 5 (36%) points, respectively. Areas III and VI had a mean of 2 sites both (14%) and areas I and IV, one (7%).

Conclusion

In accordance with the clinical literature, the branches of the temporal nerve in the temporal muscle corresponded to the described areas of the myofascial trigger points (MTPs). Anatomical correlations between MTPs may be a useful tool for a better understanding of the physiopathology of these disorders and may provide a rational basis for their treatment.
Functional Balance among Middle‐aged Adults is a Risk Factor for Cardiovascular Disease
Authors: Twiddy Hannah M. , Wilson Patrick B. , Reynolds Leryn J.
Published: May 2022

Vascular diseases are associated with falls in older adults (>65 years), but this relationship is not well understood in middle‐aged adults. This study aimed to determine if balance in middle‐aged adults with poor ankle brachial pressure index (ABPI) was impaired compared to those with normal ABPI. We hypothesized middle‐aged adults with vascular disease, measured through ABPI, will fail the Romberg Balance Test at a higher rate compared to those without vascular disease. Data was collected from the 1999‐2000 and 2001‐2002 National Health and Nutrition Examination Survey. Good balance was determined by passing the 4th condition of the Romberg Balance Test; failure wad defined as not passing the 4th condition or any of the preceding conditions. Poor ABPI was classified as ≤0.90 and normal ABPI as >0.90. Middle‐aged adults (50.1 ± 0.23) with poor ABPI have a higher frequency of poor balance (4.2%) compared to good balance (1.3%; p=0.001). Further, individuals with poor ABPI have 3.38 (CI: 1.66, 6.87; p=0.001) times the odds of having poor balance compared to individuals with normal RABPI, which remained significant after adjusting for age and body mass index. Individuals who are at a greater risk for cardiovascular disease, as assessed by ABPI, have higher odds of poor balance. Thus, poor balance appears to be a characteristic of middle‐aged individuals at risk for cardiovascular disease.
Contrasting Effects of Age on Muscle Contractility in Male and Female Rats
Authors: Russ David W. , Sehested Courtney , Weisleder Noah , Deschenes Michael R.
Published: May 2022

This study aimed to describe changes in forelimb grip strength and ex vivo muscle force production in adult (Ad) and aged (Ag, 75% lifespan) male (M) and female (F) F344/BN hybrid rats. We hypothesized that differences in M vs. F would be explained by muscle mass, but that age differences would persist after normalizing to mass in fast, but not slow muscles. Forelimb grip was expressed in absolute terms (Grip) and relative to body mass (NGrip). Ex vivo forces of the extensor digitorum longus (EDL) and Soleus (Sol) were assessed at 3 stimulation frequencies and 2 pulse durations, and expressed as absolute muscle force (AMF) and muscle quality (MQ; force/anatomical cross‐sectional area). We also assessed sarcoplasmic reticulum (SR) Ca2+ release and reuptake from homogenates of both muscles. Body mass was higher in Ag vs. Ad and M vs. F (both p < 0.001), while EDL and Sol mass were smaller in F vs. M (p < 0.001), with no effect of age. Grip showed no effect of age, but was significantly (p < 0.001) lower in F while NGrip was higher in Ad vs. Ag (p < 0.001) and F vs. M (p < 0.001). EDL AMF exhibited a significant age X sex interaction (p = 0.006) across frequencies, as AMF declined with age in M, but increased in F. EDL MQ showed a similar pattern (p = 0.039), but only at the highest frequency (120Hz). In Sol, M showed a trend (p = 0.078) for higher AMF relative to F. In contrast, MQ was significantly higher in F vs. M (p = 0.025). Differences in SR function did not appear to contribute to these differences as the only significant effect observed was for SR Ca2+ uptake to be reduced in F vs. M in both EDL and Sol (p = 0.046 & 0.050, respectively). These findings indicate that grip and isolated muscle contractility are generally higher in M vs. F when expressed in absolute terms, but lower in M vs. F when expressed as relative measures (normalized to body mass or muscle size). Minimal declines in muscle contractility occurred with age in either muscle tested, consistent with what we have observed previously with in situ testing of the Sol, but other aged fast muscles.
Development of a Specific 5‐HT7 Receptor Antibody: Is This Even Possible?
Authors: Tragge Will , Flood Emma , Lisabeth Erika , Schulz Stefan , Watts Stephanie W.
Published: May 2022

Our laboratory has a vested interest in measuring the location and expression of the 5‐hydroxytryptamine (5‐HT, serotonin) 7 (5‐HT7) receptor. The 5‐HT7 receptor is responsible for the fall in blood pressure when 5‐HT is infused acutely or chronically in the rat. Determining tissue‐specific receptor expression would aid in understanding mechanisms that support the 5‐HT7 receptor‐mediated fall in blood pressure. We have, using tissues from a 5‐HT7 receptor KO rat we created, tested over a dozen commercially available 5‐HT7 receptor antibodies. None can discriminate between WT and KO tissues, indicating a lack of specificity that has been found commonly with antibodies directed against G protein coupled receptors (GPCRs), of which the 5‐HT7 receptor is one. We contracted with 7TM antibodies (Germany) to develop rigorously a 5‐HT7 receptor specific antibody, knowing there was a 50% chance of failure. Three different antigens, two targeting the third internal loop and one the C terminus, were used in rabbits to generate antibodies. We sent 7TM a plasmid for the r5‐HT7 receptor which, transiently expressed in HEK293 cells, served as a positive control for screening initial antibodies. In Western analyses done in Germany, nine (9) antibodies (all antigens represented) detected a ~75 kDa protein that was not present in homogenates of non‐transfected HEK293 cells. These same antibodies were sent to MSU for testing in the following ways: analyses of r5‐HT7 receptor expressing HEK293 cells to demonstrate protein dependence of antibody binding in Westerns; immunohistochemical analyses of brains from 5‐HT7 receptor WT and KO rats. Only antibodies that recognized the C terminus of the 5‐HT₇ receptor [ERPERSEFVLQNSDH(Abu)GKKGHDT] positively and concentration‐dependently identified the r5‐HT7 receptor expressing HEK293 cells in Westerns. By contrast, these same C‐terminus antibodies have not specifically detected the 5‐HT7receptor in rat tissues. No specificity of signal was observed between brain sections of WT and KO rats when tested immunohistochemically. Future studies will explore different protein isolation and immunoprecipitation in buffers specialized for GPCR extraction with the goal of increased specificity of antibody signal.
A Standardized, Scalable Method to Quantify in Vitro Invasiveness
Authors: Scahill Steven D. , Sherman Kelly J. , Nichols Charles D. , Gould Harry J. , Paul Dennis
Published: May 2022

In vitro invasiveness is a fundamental measure of metastatic capacity of cells grown in culture, as the ability of a cell to permeate a semi‐solid matrix is perhaps the best indicator of its potential to invade tissue layers in vivo. However, there are significant drawbacks to current methodologies for studying in vitro invasiveness. Collagen or Matrigel provide physiologically relevant substrates for an invasive barrier, but they are costly, susceptible to batch variance, and require cold storage and minimal handling at room temperature. Transwell membranes and compartmentalized microscope slides are commonly used platforms to measure cell invasion, but these are also costly and labor‐intensive to set up and quantify. An ideal invasiveness assay would be inexpensive, user‐friendly, replicable, and scalable. To this end, we developed a method that uses standard tissue culture plate in which we form a matrix of agarose solution mixed with supplemented culture medium. Wells are made in the gel with a serological pipette, and cells are seeded into the well. The cells permeate the surrounding gel matrix over seven days, at which point the cells are fixed with paraformaldehyde solution. The area of cell invasion into the gel is the measured output of the assay. This can be measured manually, by marking the invasive edge in four cardinal positions, measuring two diameters with a caliper and calculating the invasive area. The quantification can also be automated by staining the gel with cresyl violet, imaging with a standard camera and quantifying the invasive area with a simple Image J code, or by staining the cells with 4′,6‐diamidino‐2‐phenylindole (DAPI) or a similar fluorescent nuclear label and measuring the invasive area with a blot imager. The results correlate with existing methods to quantify invasiveness while being far less labor‐intensive and costly. In summary, this method to quantify cell invasion is simple, reliable, and feasible for any researcher with basic tissue culture supplies. The method also provides quantifiable data that can be collected manually or automated and scaled to fit a wide range of needs.
Computational and Genetic Exploration of RNA‐binding FinO‐domain Structures
Authors: Dailey Katherine , Wang Suxuan , Stockert Oliver M. , Pandey Smriti , Wang Shiying , Berry Katherine E.
Published: May 2022

Evolving research on small RNAs (sRNAs) in bacteria implicates sRNAs as a key effector of gene regulation. While some sRNAs are able to act independently, many are dependent on an RNA‐binding protein, such as the well‐established Hfq in Escherichia coli. Another family of RNA‐binding proteins is the FinO family, including ProQ and FinO in E. coli, NMB1681 in N. meningitidis, and Lpp1663 in L. pneumophila. Structures for these proteins have been solved through both NMR and X‐ray diffraction, in addition to computational predictions. While many structural elements are common across all structures, there are interesting differences in regions that have been implicated by genetic experiments to be important for RNA binding. In order to investigate the structure and function relationships of these proteins, we have analyzed the available models for FinO family proteins to compare intriguing structural features, including the position and predicted contacts of a universally conserved arginine that plays a critical role in RNA binding. Finally, we are probing predicted interactions from structural models with the use of site‐directed mutagenesis and our laboratory’s bacterial three‐hybrid (B3H) assay. Together, this work is generating insights into the most relevant structural conformations for in vivo RNA binding by FinO proteins and the ways in which the structure of E. coli ProQ is both similar and distinct from orthologous FinO domain proteins.
Bi‐directional pH dependency of CALHM1 ion channel
Authors: Kwon Jae Won , Jeon Young Keul , Kim Sung Joon
Published: May 2022

Calcium homeostasis modulator (CALHM) membrane proteins are nonselective ion channels drawing attention regarding their roles in modulating neuronal activity and taste sensation. CALHM1‐expressing cells show voltage‐gated slowly activating ionic currents. The voltage‐dependence of CALHM1 is negatively affected by extracellular Ca2+ ([Ca2+]e), while facilitated by temperature. Here we investigated the effect of extracellular and intracellular pH (pHe and pHi) on the electrophysiological properties of CALHM1 overexpressed in CHO cells. When normalized to the control at pHe 7.4, the amplitudes of CALHM1 current (ICALHM1) were suppressed to 20 % at pHe 6.2, while increased to 500 % at pHe 8.6. In the whole‐cell configuration of patch clamp, changing pHi from 7.4 to 6.2 or 8.6 showed the acid‐induced inhibition and alkali‐induced activation of ICALHM1. The voltage‐dependence of CALHM1 was shifted to the negative membrane potential at the alkaline pHe and pHi, while positive membrane potential at the acidic pHe and pHi. The pHi effect was also confirmed in the inside‐out configuration of patch clamp. The open probability (Po) of multiple CALHM1 channel decreased at pH 6.2, while increased at pH 8.6 compared with pH 7.4. To get a clue to identify the pH sensing amino acid residues, we utilized homology model performed with the SWISS‐MODEL workspace, based on the cryo‐EM structure of human CALHM2. Then we conducted site‐directed mutagenesis of the water‐accessible charged amino acids of CALHM1. Among 23 candidates, mutations at E17, K229, E233, D257 and E259 in the intracellular space reduced the alkali‐induced facilitation of ICALHM1, while not altered the acid‐induced inhibition. According to the molecular structure model of killifish CALHM1 with octameric structure, the five residues located at N or C termini gathered closely in the intracellular space. Considering the large pore diameter of CALHM1 (~14 Å), we cautiously propose that the sensitivity to alkaline pHe could be because the protons transfer between hydronium ions in the pore. The study firstly demonstrated the remarkable pH‐sensitivity of CALHM1 and its pH‐sensing residues, which might be responsible for the modulation of neuronal excitability during dynamic changes of the regional pH in brain or sensory organs.
Tunnel Shape of Aldehyde Deformylating Oxygenase Determines Length of Alkane Products
Authors: Yazdani Syed Shams , Siddique Mansoor , Gupta Mayank
Published: May 2022

R&D on renewable energy has gained the centre stage again due to renewed call to tackle climate change. In this effort, drop‐in fuels like alkane will be the main biofuel candidate since they are similar to the petroleum fuel in their chemical and physical properties. Cyanobacterial AAR (acyl‐ACP reductase) ‐ ADO (aldehyde deformylating oxygenase) pathway, which converts fatty acyl‐ACP intermediate of the fatty acid synthesis cycle into alkane/alkene via an aldehyde intermediate, was the first pathway discovered for the microbial production of hydrocarbon. We have earlier reported an engineered thermostable ADO and demonstrated metabolic modelling based pathway engineering in E. coli to produce highest alkane titer reported so far. We have further performed biochemical characterization of ADOs from three different cyanobacteria (Nostoc punctiformes, Oscillatoria jasorensis, Oscillatoria sp CCC305), and found out that ADO from N. punctiforme(NpADO) yielded maximum alkane titer, with preference towards C12‐chain length aldehyde substrate. Upon structural modelling and molecular dynamics (MD) simulation, it was found that NpADO has suitable tunnel shape and size to facilitate catalysis of C12‐chain substrate. Considering the importance of the catalytic products in the aviation fuel range, we attempted to further improve the product specificity by mutating residues in the main tunnel to facilitate fitting of C12‐chain substrate in the cavity and blocking other less favorable tunnel. The engineered ADO showed 2‐fold higher catalytic activity towards C12‐chain aldehyde. MD simulations of the engineered ADOs indicated favorable tunnel structure, stabilized ADO‐substrate interactions and enhanced interaction of substrate with the di‐iron centre. These findings suggest ADO tunnels play a critical role in defining their substrate specificity and catalysis.
Genome‐wide Association Study of Oxidative Phosphorylation in Cocks Semen after Freezing
Authors: Nikitkina Elena , Musidray Artrem , Dementieva Natalia , Pleshanov Nikolai , Shcherbakov Yuri , Plemyashov Kirill
Published: May 2022

The biochemical processes associated with the generation of energy (ATP) ‐ oxidative phosphorylation (OXPHOS) and glycolysis ‐ occupy a central place in the vital activity of cells. OXPHOS is the terminal process of cellular respiration (CR). Aim of the study was Genome‐wide Association Study of OXPHOS in cocks sperm after freezing. Semen from 96 cocks was frozen. CR was assessed by the polarographic method. OXPHOS can be evaluated by the reaction CR on adding 2,4‐dinitrophenol (2,4 ‐ DNP) to semen sample. 2,4 ‐ DNP disrupt the proton gradient by carrying protons across a membrane and uncouples proton pumping from ATP synthesis because it carries protons across the inner mitochondrial membrane. As a result it increases respiration rate. If the respiration rate increases – there is good OXPHOS. Sperm motility was evaluated by computer‐assisted semen analysis (CASA) (ArgusSoft, Russia) before and after freezing. The evaluation was carried out using an Axio Imager microscope (Carl Zeiss Microscopy GmbH, Germany). DNA was isolated from sperm samples for genotyping on Gallus Gallus – Affymetrix Axiom®600k Array. Calculations were performed using the PLINK and EMMAX programs. The Genome‐Wide Association Studies (GWAS) was used for the analysis of genomic associations. Results are depicted as ranges, percentages of totals and averages ± SEs. The volume ejaculate varied from 0.4 ml to 1.4 ml, motility before freezing was from 20 to 95% and motility after freezing was from 5 to 80%. The 2.4‐DNP (FSRA) stimulation of respiration after freezing ranged from 1 to 8.09, averaging 1.66±0.114. The high repeatability of the FSRA score across samples suggests the presence of genetic factors, the identification of which can serve as a test for selecting animals with sperm whose quality remains fertile after freezing. 28 potential SNPs associated with FSRA the were found. На 5 хромосоме было найден также блок SNPs (большинство из них significant, 4 последних ‐ suggestive: AX‐76791596 (P‐value=6,20E‐09), AX‐76791535 (P‐value=1,83E‐08), AX‐76791611 (P‐value=1,83E‐08), AX‐76791454 (P‐value=1,87E‐08), AX‐76791629 P‐value=2,11E‐08), AX‐76791570 (P‐value=4,04E‐08), AX‐76791651 (P‐value=5,43E‐08), AX‐76791640 (P‐value=8,84E‐08), AX‐76791553 (P‐value=9,94E‐08), AX‐76791503 (P‐value=1,18E‐07), AX‐76791443 (P‐value=5,49E‐07), AX‐76791439 (P‐value=1,41E‐06), AX‐76791501 (P‐value=1,63E‐06). The significant SNPs found were located in the RAG2 gene. We suggest that the polymorphism in this gene is associated with the selectivity of fertilization, and the efficiency the work is associated with ATP‐dependent remodeling of ATPase subunits. Studies have opened up the possibility of studying the changes in the cells after freezing. Found SNPs markers will be used to select males whose semen is suitable for cryopreservation.
Resonance Raman of the nitrite reductase action of heme‐copper oxidoreductases
Authors: Varotsis Constantinos , Tselios Charalambos , Pinakoulaki Eftychia
Published: May 2022

Several themes have emerged in the studies of biological control of nitrite and nitric‐oxide chemistry.[1‐2] Apparently, evolution has clearly selected means by which to take advantage of the unique NO2 and NO chemistry, and a number of enzymes have evolved that are able to activate NO2/NO kinetically, and control their redox chemistry. In bacterial respiration the reduction of NO2/NO to N2O by terminal oxidoreductases supports the hypothesis of a common evolutionary origin of bacterial denitrification and aerobic respiration. Comparison of the enzymes responsible for the activation of NO in denitrification and bacterial respiration may provide the means to identify conserved structural features, which can be assumed to be involved in basic functions common to both classes of enzymes. The most commonly encountered metals in the biological reduction of NO2 to NO and subsequently to N2O are iron and copper in mononuclear, binuclear, and dinuclear centers. In P450Nor the electrons needed for the reduction of NO are directly transferred from NADH. This way, a single molecule of NO binds at the heme Fe, and addition of two electrons to heme Fe3+‐NO yields the two electron reduced species Fe2+‐N=O‐. A second NO moleculeattacks the N atom of the ferrous‐NO species to transiently yield hyponitrite (HONNO–), and thus the N‐N bond formation. Cleavage of the N‐O bond produces the ferric enzyme, N2O and H2O. Resonance Raman studies of the NO2‐ligand binding properties of heme‐copper oxidases will be presented.

References.

Discrete Ligand Binding and Electron Transfer Properties of ba3‐Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and …C Koutsoupakis, T Soulimane, C. Varotsis Accounts of chemical research 52 (5), 1380‐139, 2019.

The structure of a ferrous heme‐nitro species in the binuclear heme a3/CuBcenter of ba3‐cytochrome c oxidase as determined by resonance Raman spectroscopy A. Loullis, MR Noor, T Soulimane, E Pinakoulaki Chemical Communications 51 (2), 286‐289, 2015.
Reproducibility of Respiratory Chemoreflex Characterization by Modified Rebreathing
Authors: Huggard Joshua D. , Guluzade Nasimi A. , Keir Daniel A. , Keltz Randi R.
Published: May 2022

Modified hypoxic and hyperoxic rebreathing is used to characterize central and peripheral respiratory chemoreflexes in humans. This study examined within‐ and between‐day reproducibility of the key parameters that are identified by this method: ventilatory recruitment threshold (VRT) and chemoreflex sensitivity. Seven healthy males (mean ± SD; age: 27±5 years) visited the laboratory on 4 occasions to performrepetitions of modified rebreathing tests in isoxic‐hypoxic (end‐tidal PO2 (PETO2) =50 mmHg) and ‐hyperoxic conditions (PETO2=150 mmHg). Days 1 and 2 consisted of three repeatedhyperoxic and three hypoxic trials, respectively; Days 3 and 4 included both a hyperoxic and a hypoxic trial completed in a counterbalanced order. Ventilation, end‐tidal PCO2 (PETCO2) and PETO2 were measured breath‐by‐breath by pneumotach and dual gas analyser. For each rebreathing trial, the PETCO2 at which ventilation began to rise was identified as the VRT (mmHg) and the slope of the response above VRT provided respiratory chemoreflex sensitivity (L∙min‐1∙mmHg‐1). Within‐ and between‐day reproducibility was assessed by one‐way ANOVA and intraclass correlation (ICC) analyses. The VRT was not different between trials performed on a single day for bothhyperoxic (44±2, 45±3, vs 44±3 mmHg; for Trials 1, 2, vs 3, respectively; p=0.50; ICC=0.926) and hypoxic rebreathing (40±3, 40±4, vs 40±4 mmHg; p=0.66; ICC=0.994). Chemoreflex sensitivity also was not different between trials performed on the same day in hyperoxic (4.3±2.2, 5.0±3.1, vs4.8±2.3 L∙min‐1∙mmHg‐1; p=0.33; ICC=0.955) or hypoxic conditions (5.6±2.2, 6.2±2.7, 5.7±2.0 L∙min‐1∙mmHg‐1; p=0.33; ICC=0.953). Between days, the VRT was not different for hyperoxic rebreathing (44±3 vs 44±2 vs 45±2 mmHg for Days 1, 3, vs 4, respectively; p=0.10; ICC=0.813) but was different for hypoxic rebreathing (40±3 vs 40±2 vs 41±3 mmHg for Days 2‐4, respectively; p=0.01; ICC=0.931). Chemoreflex sensitivity was not different between days for either hyperoxic (4.8±2.5,4.4±2.7, vs 4.9±2.0 L∙min‐1∙mmHg‐1; p=0.01; ICC=0.906) or hypoxic rebreathing (5.8±2.2, 5.8±2.6, vs 7.8±2.8 L∙min‐1∙mmHg‐1; p=0.01; ICC=0.750). Collectively, these data indicate that model parameters derived from hyperoxic and hypoxic modified rebreathing have good‐to‐excellent test‐retest reproducibility when repeated on the same or different days.
AJAP1 Gene Silencing Alters Protein Expression in Epithelial Cells
Authors: Gerick Alexander , Osuji Nnedi , Jeffers Cameron , Hatcher Cathy J.
Published: May 2022

Introduction

The epicardial layer of the heart is formed during cardiovascular development from a transitory structure known as the proepicardium (PE). Proepicardial cells migrate out of the PE to surround and adhere to the myocardium to form a layer of epithelial cells that compose the epicardium. A subset of the epicardial cells delaminate and migrate into the subepicardial space where they undergo epithelial‐mesenchymal transition (EMT) to become epicardial‐derived cells (EPDCs). These EPDCs invade the myocardium and differentiate into a subset of cells that make up the coronary vessels. During this entire process, the epicardium receives input from several transcription factors including Tbx5. Mice with a conditional deletion of Tbx5 exhibit impaired epicardium formation and coronary vessel development. Tbx5‐deficient mouse hearts have a significant decrease in mRNA expression of adherens junction associated protein (Ajap1). Our studies show that knockdown of endogenous AJAP1 (AJAP1 KD) mRNA expression in epithelial cells leads to increased cell adhesion, reduced cell migration, enhanced expression of epithelial markers of EMT, and altered expression of transcripts associated with adherens junctions, tight junctions and EMT. Specifically, our qPCR array data shows a significant alteration in ZEB2 mRNA expression in AJAP1 KD cells versus negative control cells.

Study Objective

The goal of this study was to examine the role of AJAP1 in regulating protein expression of ZEB2 as it may be necessary for epicardium formation. Based on previous cell function and mRNA expression data, we hypothesize that the ZEB2 protein is an important functional mediator of AJAP1 in epithelial cells for undergoing EMT.

Methods

We utilized the primary human mammary epithelial cell line (HMEpiC) as an in vitro system to examine contributions of AJAP1 to epithelial cell biology. We silenced AJAP1 mRNA expression in HMEpiCs using small interfering RNAs and examined protein expression of ZEB2. We compared ZEB2 expression in negative control versus AJAP1 KD HMEpiCs.

Results

Reduced expression of AJAP1transcripts produced noticeable changes in protein expression of ZEB2 in epithelial cells, and it had significant effects on expression of adherens junction‐related transcripts. Western blot analysis revealed an increase in ZEB2 protein expression in AJAP1 KD versus negative control HMEpiCs.

Conclusion

Our data indicate that AJAP1 contributes to epithelial cell function, and it does this potentially by mediating ZEB2 protein expression. ZEB2 is known to regulate EMT, which is a biological process that is crucial for the formation of the epicardium and its subsequent formation of the coronary vasculature.
Increase in Salt Concentration in the Culture Media Enhances Protein Expression of Tumor Necrosis Factor‐Alpha Receptor Type 1 in Cultured Renal Cortical Collecting Duct Cells But Not in Proximal Tubular Cells
Authors: Majid Dewan S. A. , Castillo Alexander
Published: May 2022

Tumor necrosis factor‐alpha (TNFα) exerts its biological responses via interaction with its’ two cell surface receptors, type 1 (TNFR1) and type 2 (TNFR2) which are differentially expressed and regulated in the kidney. Previously, we have demonstrated that circulating TNFα exerts natriuresis via its action on TNFR1 but not on TNFR2 and such TNFR1 activation exerts inhibitory action on distal tubular sodium transport, particularly tubular ENaC activity. As TNFα level is elevated during dietary intake of high salt (HS), we hypothesized that increases in salt concentration in renal tissue enhances TNFα ‐TNFR1 activity in the renal tubules to induce natriuretic response to HS intake. To test this hypothesis, we examined the protein expression of TNFR1 and TNFR2 in cultured proximal (human kidney [HK‐2] cell line) and distal (murine cortical collecting duct [M‐1] cell line) tubular cells at different concentrations of NaCl in the culture media. Both these HK‐2 and M‐1 cells were incubated for 24 hours in control media with normal NaCl concentration (120 mM) and at media containing higher NaCl (140 mM and 160 mM) concentrations. TNF receptors protein expressions were analyzed by the Western Blot technique using appropriate antibodies for TNFR1 (rabbit anti‐TNFR1; Abcam cat # 19139) and TNFR2 (rabbit anti‐TNFR2; Abcam cat # 109322), which were normalized by expression of β‐actin or GAPDH proteins using appropriate antibodies. The blot image was quantified and measured as band intensity density with Image J software. The quantitative immunoblot analysis in HK‐2 cells showed that there were no significant changes in the band intensity unit (IU) in TNFR1 (120 mM, 70.1±12.8 IU; 140 mM, 73.7±9.9 IU; 160 mM, 57.2±9.8 IU; n=6 in each condition) or TNFR2 protein (120 mM, 83.9 ±6.3 IU; 140 mM, 66.8±14.1 IU; 160 mM, 77.8±2.3 IU; n=6 in each condition) levels. On the other hand, TNFR1 protein expression was significantly higher in M‐1 cells incubated in 160 mM NaCl conc. (135.6 ±7.8 IU, n=7; P< 0.005) than that in 120 mM (109.3±2.8 IU, n=7) and in 140 mM (91.1±10.8 IU, n=7) NaCl conc. However, TNFR2 protein expression in M‐1 cells did not significantly alter with the changes in NaCl conc. in the media (120 mM, 137±20.1 IU; 140 mM, 128±7.2 IU; 160 mM, 143±7.2 IU; n=7 in each condition). These data indicate that TNFR1 activity is upregulated in renal collecting duct cells (CCD) in response increases in tissue NaCl concentration in the kidney. Such upregulation of TNFR1 activity in the CCD induced by high salt concentration, exerts inhibitory action on distal sodium transport and plays a role in enhancing urinary sodium excretion during dietary HS intake.