bluuebunny/crossref_metadata_2025 huggingface dataset:
O‐GlcNAc characterization during Tribolium castaneumdevelopment
Authors: da Costa Rodrigues Bruno , Gomes Neto Lupis R. , Todeschini Adriane R. , Dias Wagner B. , da Fonseca Rodrigo N.
Published: May 2022

O‐GlcNAc (O‐linked β‐N‐acetylglucosamine) is a dynamic intracellular post‐translational modification (PTM) that regulates several cellular processes. This PTM is catalyzed by the covalent attachment of GlcNAc on residues of serine/threonine in proteins by the enzyme O‐GlcNAc transferase (OGT), while its removal is performed by O‐GlcNAcase (OGA). This PTM is limited by the concentration of UDP‐GlcNAc, the end‐product from the hexosamine biosynthetic pathway (HBP) that, together with OGT and OGA levels and activity, will regulate protein O‐GlcNAcylation. Although O‐GlcNAc has been reported in all studied metazoans, functional analysis during development in Hexapoda is restricted to the model organism Drosophila melanogaster. In this study, the role of O‐GlcNAc during development was investigated in an emerging model system, the beetle Tribolium castaneum. Comparison of the transcriptional profile of the HBP rate‐limiting step enzyme glutamine‐6‐phosphate amidotransferase (GFAT) during developmental stages showed higher levels during larval stages. Interestingly, OGT is modulated during embryonic development with an increase of mRNA levels during gastrulation while OGA remained unaltered in all stages. In situ hybridization analysis revealed an embryonic limited ubiquitous pattern of OGA during egg development, while GFAT is expressed in both embryonic and extra‐embryonic cells. Finally, parental knock down of OGT by RNA interference (RNAi) shows physiological impairment of oocyte maturation and a decrease in egg laying and embryonic survival, while OGA knockdown did not lead to physiologically and phenotypic significant changes. Altogehter, these results provide an important characterization of the O‐GlcNAc machinery in the most diverse order of insects, and suggest a critical role of O‐GlcNAcylation in a stage‐specific manner during Tribolium castaneum development.
Understanding of the Serotonin Transporter Mechanism‐Targeting Drug Therapy
Authors: Fassler Amy , Rens Davis , Shah Ruhi , Chojnacki Kennan , Rens Kai
Published: May 2022

Depression and anxiety are ranked in the top five of causes of disabilities worldwide and are the most prevalent psychiatric disorders. Serotonin is a vital hormone that commonly acts as a neurotransmitter to modulate brain activity and enable communication between nervous system cells. The serotonin transporter (SERT) is a protein located within the presynaptic membrane and is activated by 5‐hydroxytryptamine (5‐HT), a chemical messenger that releases serotonin. The SERT protein re‐uptakes 5‐HT during its release, leading to the regulation of the neurotransmitter’s concentration in the synaptic cleft. Over‐activation of SERT leads to less 5‐HT, in turn lowering serotonin concentration, resulting in a higher chance of developing depression. Common drug treatments such as citalopram and paroxetine then bind at the central site, creating an outward‐open conformation. The central site of this protein is located halfway across the membrane, wedged into a cavity made up of residues from transmembrane helices. This new structure exposes a cone‐shaped extracellular vestibular that provides an open pathway to the central site. This process does not directly inhibit the serotonin transporter, but instead allosterically opens the Thr497‐Phe335 motif and creates a new binding pathway called S2. The opened S2 allows for new chemical binding that will inhibit SERT and prevent re‐uptake. Our research attempts to explore how various antidepressants prevent the re‐uptake of 5‐HT by inhibiting this serotonin transporter.
Characterization of the spontaneous right ventricular hypertrophy and evolution to heart failure in Wistar Kyoto rat strain
Authors: Cortes Gallardo Ernesto , Navarrete Peñaloza Grecia , Bocanegra Alfaro Ana , Pérez López Roberto , Gómez Villalobos Ma de Jesus
Published: May 2022

Heart failure is a pathological condition that remains as a major cause of morbi‐mortality in the world, and several cellular mechanisms involved in the development of heart failure remain unknown. In this study it was characterized a spontaneous experimental model of right ventricular hypertrophy from Wistar Kyoto rat strain and monocrotaline injection as a factor to induce the development of heart failure. Morphological and functional cardiovascular parameters, and atrial natriuretic factor (ANF) in serum were measured to determine criteria to be considered as a model of heart failure. Male WK rats 2 months old were assigned to 3 experimental groups (n=8 each group): control, right ventricular hypertrophy + saline (RVH+S) and right ventricular hypertrophy + monocrotaline (RVH+MCT). RVH was diagnosed through no invasive electrocardiographic record in awake animals, and one dose of monocrotaline (60 mg/Kg, ip) was injected. After 3 weeks, cardiovascular parameters were recorded (systemic and pulmonary systolic arterial blood pressure, heart rate), and arterial blood from aorta was taken and the heart was removed and analyzed. Total heart, right ventricle, left ventricle weights (g), and right ventricle free wall (in mm) were significantly different in the RVH+S (1.3±0.02, 0.40±14.11, 0.58±13, 2.86±0.19) and the RVH+MCT (1.6±0.09, 0.53±17.5, 0.67±18.94, 4±0.32) groups in comparison with the control group (0.99±0.04, 0.22±4.0, 0.55±14.38, 2.13±0.05), P>0.001 for all parameters, except for LV weight in the RVH+S group. The systemic arterial blood pressure analysis demonstrates absence of significant difference between groups, however, right ventricular pressure (RVP in mm Hg) in the RVH+S (42±2.53), and RVH+MCT (78±4.22), was significantly increased in comparison with the control group (22±1.16), P<0.001. ANF levels in serum were higher in the RVH+S and RVH+MCT experimental groups in comparison with the control group (537±11.04, 557±7.7 and 473±10.65 in pg/ml, respectively; P<0.01). The results suggest that a sub‐strain from WK rats develop right ventricular hypertrophy spontaneously and with the administration of monocrotaline, severe right and left ventricular hypertrophy was developed, changes associated to an increase in levels of atrial natriuretic peptide suggesting heart failure, then could be a useful experimental model of heart failure where right ventricular hypertrophy is a central pathologic mechanism.
Correlation of Cardiovascular Function and Hemodynamic Parameters with Gender and BMI in Healthy Subjects
Authors: Zhang Yanfang , Sun Xinguo , Wang Jinan , Zhou Qingqing , Tai Wenqi
Published: May 2022

Objective

To observe and study the left heart function and hemodynamic parameters in young healthy normal subjects, and to analyze the correlation between gender and body mass index (BMI) and left heart function.

Methods

We selected thirty‐nine young healthy graduate students, advanced training doctors, and visiting scholars from Fuwai Hospital without any disease diagnosis and low daily exercise. Hemodynamic parameters were collected by using a non‐invasive synchronous ventricular function detector (N‐ISVFD). We calculated the abnormal rate of hemodynamic parameters and analyzed the correlation between gender and BMI and left heart function.

Results

In this study,39 healthy young subjects (males 18, females 21), aged (30.1±5.6) years old, BMI(22.0±2.9) kg/m2,all participants signed the written informed consent. The abnormal rate of left ventricular jet pressure (LVJP), systolic mean pressure (SMP), diastolic mean pressure (DMP), left ventricular end diastolic volume (LVEDV), myocardial oxygen consumption (MOC), ejection fraction (EF), left cardiac index (LCI) were 39.2%±22.6%. LVJP, SMP, DMP, LVEDV and MOC were correlated with gender and BMI. EF and LCI were relatively independent of gender and BMI.

Conclusion

Due to the stress life, psychological and other aspects, there is no symptoms or various checks presented negative, hemodynamic parameters of young people has deviated from the normal range. We should take more exercise to prevent diseases from happening. Indicators in connection with myocardial contractility, left ventricular preload, left ventricular afterload and coronary circulation are related to gender and BMI, so the assessment of cardiovascular function of patients should take into account gender and BMI differences.
Targeting Adipose Tissues with ATS9R Nanoparticles for Drug Delivery
Authors: Orr Alexis W. , Liu Kunli , Huang Xuefei , Watts Stephanie W.
Published: May 2022

Nanoparticles (NPs) are a drug delivery system that can enable delivery of a drug to a targeted tissue. Previous studies have shown that the use of an adipose targeting sequence (ATS) peptide in conjunction with a drug in the form of a NP has the ability to selectively deliver the drug to mouse adipose tissues. The prohibitin protein located in adipose tissue is the target of ATS. Adipose tissue is a major source of the adipokine chemerin, a prohypertensive protein. Our laboratory has proven that liver derived chemerin, the largest source of circulating chemerin, is biologically inactive. Our goal is to understand if chemerin produced by adipose tissue contributes to hypertension. We hypothesize an ATS drug delivery system could be applied in rats to specifically reduce the levels of adipose tissue associated chemerin. We created a NP consisting of an antisense oligonucleotide (ASO) against chemerin and a FITC labeled ATS with 9 arginine sequence (ATS9R). In vitro studies showed that the ASO is functional when incorporated into NPs with ATS9R as it reduced chemerin mRNA expression in isolated epidydimal (Epi) adipocytes by 43‐fold compared to vehicle incubated cells (Veh mean 2‐ΔCT=0.039 ± 0.007; NP mean 2‐ΔCT=0.0009 ± 0.0001), and 13‐fold in isolated retroperitoneal (RP) adipocytes (Veh mean 2‐ΔCT=0.041 ± 0.007; NP mean 2‐ΔCT=0.0031 ± 0.0009) of Dahl SS rats. Ex vivo studies support the ability of the ATS9R‐ASO NP to reduce chemerin mRNA expression in tissues. In Epi fat incubated with the NP overnight at 37 oC, chemerin expression was reduced by 2.7‐fold (p<0.05) (Veh mean 2‐ΔCT=0.213 ± 0.088; NP mean 2‐ΔCT=0.079 ± 0.013). The NP also reduced chemerin expression in RP fat by 2.1‐fold (Veh mean 2‐ΔCT=0.159 ± 0.027; NP mean 2‐ΔCT=0.071 ± 0.011), although the changes did not reach statistical significance. However, this same NP was unable to deliver the ASO selectively to adipose tissue in vivo. Two days after subcutaneous injections in Dahl SS rats, the NP caused a significant (p<0.05) 2.4‐fold reduction in liver chemerin mRNA expression compared to vehicle treated animals (Veh 2‐ΔCT=0.753; NP mean 2‐ΔCT=0.311 ± 0.007), but not in Epi (1.8‐fold reduction; Veh 2‐ΔCT=0.175; NP mean 2‐ΔCT=0.095 ± 0.013) or RP fat (1.9‐fold reduction; Veh 2‐ΔCT=0.120; NP mean 2‐ΔCT=0.061 ± 0.015). Varying NP dose, administration route, and concentration of the components constituting the NP did not show any improvement in ASO delivery to fats vs liver. Further studies are needed to develop the ATS9R system to deliver ASO to adipose tissue beds in rats.
Restoration of tidal volume after spinal cord injury by hyperbaric oxygen therapy requires both pressure and 100% oxygen
Authors: Sunshine Michael , Bindi Victoria E. , Nguyen Branden L. , Doerr Vivian , Boeno Franccesco P. , Chandran Vijayendran , Smuder Ashley J. , Fuller David D.
Published: May 2022

Approximately 13,000 spinal cord injuries (SCI) occur each year in the U.S. The majority of SCIs are vertebral crush/contusion injuries at the cervical level. Cervical SCIs often result in impaired ventilation and in severe cases require full time mechanical ventilation. Hyperbaric oxygen (HBO) therapy is a promising non‐invasive treatment for SCI. By exposing the injured spinal cord to higher pressures, 2‐3 atmospheres (ATA), at increased oxygen (100%), HBO therapy helps deliver oxygen to poorly perfused tissues, has anti‐oxidant and anti‐inflammatory impact, and has been reported to mitigate neuronal loss. Additionally, HBO may mitigate muscle atrophy after SCI. Despite the promise of HBO therapy, to our knowledge there have been no studies that have comprehensively controlled for the different variables that are manipulated during HBO treatment (e.g. oxygen level, chamber pressure). In this study, we tested two hypotheses: 1) the benefit of HBO therapy after SCI requires pressurized 100% oxygen vs. either variable alone, and 2) HBO therapy can improve respiratory recovery after cervical SCI. Six experimental groups were included: one without injury (Sham injury), and five groups with a hemicontusion injury at spinal segment C4. The first injured group did not receive any treatment (SCI only), the next group was treated with 2ATA pressure, but 10.5% oxygen to target normal inspired partial pressure of oxygen. The final three groups tested the effect of 100% oxygen at 1, 2, or 3ATA. All treatments were initiated within 2 hours of injury, lasted 1‐hour, and were repeated daily for 10 consecutive days. We measured ventilation using whole body plethysmography prior to injury and on days 5 and 10 post‐injury. During normoxic conditions on day 5, all experimental groups had reduced tidal volume compared to Sham animals. By day 10, however, the tidal volume of animals in the 2ATA and 3ATA group returned to the level of the Sham animals. Further, during a period of respiratory challenge achieved by inspiring a mixed hypercapnic (7% CO2), hypoxic (10.5% O2) gas, only the animals in the 3ATA group matched the tidal volume of the Sham animals. Additionally, cross sectional analysis of the mid‐costal diaphragm revealed a preservation of the size of type IIb/x fibers in the animals in the 3ATA group. This study demonstrates that both pressure (≥2 ATA) and elevated oxygen (100%) are required to improve ventilation, and that higher pressure (3ATA) may confer greater benefits over 2ATA treatment.
Ablation of PRAK exacerbates the mitochondria dysfunction in C2C12 myoblasts exposed to palmitate stress
Authors: Wang Jianguo , Zhao Thomas , Yano Naohiro , Zhao Ting C.
Published: May 2022

Background

p38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating survival and metabolism. However, the role of PRAK in the regulation of palmitate‐induced metabolic stress remains unknown. We examined whether deletion of PRAK would suppress cell survival and mitochondrial function and whether PRAK‐induced detrimental effect was mitigated by irisin in C2C12 myoblasts in response to palmitate (PA) stress.

Methods

PRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas‐9 genome editing technology. Both wild type and PRAK‐/‐ C2C12 cells were exposed to palmitate at the concentration of 200 mmol/L for 48 h to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK‐/‐cells was determined to explore its relationship with RPAK in response to palmitate stress. Cell viability, ATP product, glucose uptake, mitochondrial membrane potential and mitochondrial swelling were assessed.

Results

PRAK knockout decreased C2C12 viability in response to PA stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to PA treatment, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential and increasing mitochondrial swelling. However, PRAK knockout induced an impairment of mitochondrial and cell damage in C2C12 cells exposed to PA stress were rescued by treatment of irisin (10ng/ml).

Conclusion

Our finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function and glucose uptake in C2C12 cells exposed to palmitate induced metabolic stress.
Enabling regeneration in a β‐catenin‐deficient liver: Unraveling a novel compensatory role of mTOR
Authors: Hu Shikai , Cao Catherine , Singh Sucha , Poddar Minakshi , Monga Satdarshan
Published: May 2022

Backgrounds

Liver can regenerate to maintain its essential functions upon injury. Several signaling pathways including the mTORC1 and Wnt/β‐catenin pathways have been shown to independently stimulate liver regeneration (LR) to promote repair. However, how one pathway could compensate during deficiency of another is less well understood. Here, we report discovery and characterization of a compensatory interplay between the mTORC1 and the Wnt/β‐catenin pathway that enables LR following liver injury when Wnt/β‐catenin pathway is interrupted genetically.

Methods

We generated hepatocyte‐specific β‐catenin knockout (β‐catenin∆HC) mice by delivering AAV8‐TBG‐Cre to 6‐week‐old Ctnnb1flox/flox mice. Two weeks after AAV8 injection, animals were either harvested at baseline, or subjected to partial hepatectomy (PH) for analysis at various time points to address changes and compensations during the process of LR. LR was also compared between β‐catenin∆HC and Albumin‐Cre‐driven β‐catenin knockouts from both hepatocytes and cholangiocytes (β‐catenin∆Liver).

Results

At baseline, a small subset of β‐catenin∆HC mice had smaller liver size and mild fibrosis, but overall, there was no impact on body weight, liver size, and liver weight to body weight ratio (LW/BW). After PH, LR was more severely delayed in β‐catenin∆HC mice compared to β‐catenin∆Liver, likely due to chronic adaptive changes that only occur in the latter. After PH, 14‐day continuous BrdU labeling through drinking water showed only 31% positively labeled hepatocytes in β‐catenin∆HC mice versus 92% positively labeled hepatocytes in controls. The scant LR in β‐catenin∆HC mice started around the periportal region at 40h and increased at 72h and peaked at 96h seen by positive staining for Cyclin D1, Ki67, BrdU (5h labeling). Western blots using whole cell lysates showed a clear increase in p‐IGF1R β‐Y1135/1136 and p‐Insulin receptor (IR) β‐Y1150/1151 at 72h, and activation of mTORC1 activity seen as increased p‐mTORC1 (S2448), p‐p70S6K (T389), p‐S6 (S235/236) and p‐S6 (S240/244) at 72h and 96h. More importantly, careful quantification showed 97% of mitotic hepatocytes to be positive for p‐S6 (S235/236) in β‐catenin∆HC. Pharmacological inhibition of IGF1R and IR by linsitinib, or inhibition of mTORC1 by rapamycin completely blocked LR in β‐catenin∆HC, further confirming that IGFR/IR‐mTORC1 pathway drove LR to ensure liver repair in β‐catenin∆HC mice.

Conclusions

Our data reaffirms an important role of β‐catenin in normal LR after PH. In the absence of β‐catenin in hepatocytes, there is spontaneous activation of IGFR/IR driven mTORC1 pathway, which in turn drives hepatocyte proliferation in β‐catenin∆HC mice. Our studies have discovered a novel axis of compensation, once again supporting existence of redundant mechanisms that enable LR when one pathway is inhibited.
A Preliminary Report on the Variation of Respiratory Blood Pressure during Sleep in Normal Subjects
Authors: Wang Jinan , Sun Xingguo , Tai Wenqi , Zhou Qingqing , Zhang Yanfang
Published: May 2022

Objective

Based on the new theory of holistic integrative physiology and medicine, we hypothesize that respiration is the origin of the variability of circulatory parameters, and initially verify this view by analyzing the relationship between blood pressure variability and respiration.

Methods

This study analyzed 8 normal subjects (NS) with no disease diagnosis who performed cardiopulmonary exercise testing (CPET), polysomnography (PSG), continuous fingertip pulse wave and electrocardiogram (ECG), and analyzed the correlation between the respiratory cycle of nasal airflow and blood pressure variation cycle during sleep. The respiratory cycle included complete inhalation and subsequent exhalation. Analysis and calculation of the number of breathing cycles (B‐n), average breathing cycle time (Bn‐T) and other parameters. Blood pressure variability is calculated using two different methods: fingertip pulse wave waveform and pulse transit time (PTT). The peak of the fingertip pulse wave represents the systolic blood pressure (SBP), and the end point represents the diastolic blood pressure (DBP). The values of the peak and end points are extracted separately and sorted separately. Continuous blood pressure rises from lowest to highest at a time, then dropped from highest to lowest, as a blood pressure variation cycle, calculation of blood pressure variation cycle (BPV‐n), and other parameters. PTT can directly calculate the blood pressure value, and it calculates the blood pressure variability in the same way as the fingertip pulse wave. To compare the relationship between respiratory and blood pressure variability parameters and the similarities and differences between the two methods, independent‐sample t test and paired‐sample t test were used for statistical test.

Results

The subject's apnea hypopnea index was 1.71±1.30 events/hour. Pulse wave waveform calculation Bn (6581.63±1411.90), SBPV‐n (6629.88±1479.84) and DBPV‐n (6628.38±1396.37) are highly consistent during sleep. Bn/SBPV‐n (1.00±0.03) and Bn/DBPV‐n (0.99±0.02) is close to 1 (P>0.05); Bn‐T (4.19±0.57 s), average systolic blood pressure variation cycle time (4.17±0.63 s) and average diastolic blood pressure variation cycle time (4.16±0.60 s) basically the same (P>0.05). SBPV‐n (3988.38±1701.93) and DBPV‐n (886.63±176.86) calculated by PTT during sleep were significantly lower than the calculated values of pulse wave waveform (P<0.01).

Conclusion

Normal subjects’ blood pressure variability during sleep is very similar to respiration, and the initiating factor of blood pressure variability is respiration.
Applications of Designed Alpha‐Helical Metallopeptides
Authors: Smith Sarah J. , Mawn Ian H. , Knight Derek W.
Published: May 2022

A variety of strategies have been employed to generate alpha‐helical peptides to mimic native protein‐protein interactions. Here, we present applications of a method that induces secondary structure in short peptides using engineered metal‐binding sites. We propose that the incorporation of metal ions to create helical peptides provides additional benefits that the more widely utilized covalent crosslinking methods lack. We have created a peptide system designed to bind ribose 5‐phosphate isomerase B (RpiB), a protein target in developing treatments for Chagas disease caused by Trypanosoma cruzi. In this system, we have compared peptide structure and stability when using our metal‐crosslinking system to hydrocarbon, lactam, and thioether staples across 1‐ or 2‐turns of an alpha‐helix. We have also measured the ability of each of these peptide constructs to bind RpiB and prevent homodimerization, which is required for parasitic activity. Further, we have studied whether metal‐bound peptides are better able to cross cellular membranes and enter the cytoplasm of mammalian cells compared to metal‐free peptides, as has been previously suggested in the literature, and deduced the impact of metal ion charge, identity, and secondary structure in enhanced permeability.
Selenoprotein S Binds to the SARS‐CoV‐2 Replication Complex
Authors: Ghelichkhani Farid , Rozovsky Sharon
Published: May 2022

The severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) uses endoplasmic reticulum membranes and associated human proteins for its replication and to evade detection. One human protein recruited by several viral proteins is selenoprotein S, which takes part in the endoplasmic reticulum protein degradation pathway, NFkB signaling, and cytokines secretion. A key interaction was reported with SARS‐CoV‐2 non‐structural protein (nsp7), which is essential for virus replication. However, it was unclear whether selenoprotein S and nsp7 interact directly and whether the interaction is possible when nsp7 forms a complex with the other components of the virus's replication machinery. We used biochemical assays to show that selenoprotein S binds nsp7, including when nsp7 is in complex with the coronavirus's RNA‐dependent RNA polymerase. This places selenoprotein S at the heart of the coronavirus's replication complex and marks it as the first human protein shown to directly interact with the viral replication complex. Cross‐linking experiments were employed to map the interactions of selenoprotein S and nsp7 in the replication complex. We show that the hydrophobic segment of selenoprotein S is essential for binding nsp7. This arrangement leaves an extended helix and the intrinsically disordered region of selenoprotein S exposed and free to recruit additional proteins to the complex.
Designer Insulins' Structural Effects on Response Time
Authors: Farhat Baaqer , Beaver Jaydon , Berry Joaquin
Published: May 2022

Insulin is a hormone that controls the level of glucose in the bloodstream. It acts as a messenger that binds to cell receptors (embedded in cell membranes), resulting in lowering sugar levels in the blood. Patients with Type 1 Diabetes mellitus lack the ability to produce Insulin which requires them to inject it to maintain glucose blood levels. There are two main types of Insulin, Human (the one that the body makes) and designer Insulin. Designer insulins differ in Beta chain amino acid sequences, which leads to a significant change in response time. This change typically takes place in B28 and B29 amino acids. It is a group interest to research how structural differences in Beta chain of Long and Short acting Designer Insulins lead to the significant change in response time compared to natural Human Insulin. Insulin Lispro (1LPH), a type of rapid/short acting Insulin with Lys28 and Pro29 beta chain configuration is effective for a duration of 5 hours. Insulin Glargine (4IYD) on the other hand, is a Long acting designer insulin with a reversed proline at B28 and lysine at B29. Galrgine is effective for 24 hours, 19 hours more than Lispro even though the difference is so small in the Beta chain of their amino acids. Human insulin has the same Beta chain configuration as Glargine, which in a no Diabatic scenario keeps glucose level balanced at all times. This reversed sequence in Lispro results in a more rapid dissolution of the Insulin, making it dimer and then to a monomer that gets absorbed rapidly straight after injection. It is also important to mention that Glargine has only 29 amino acids in the Beta chain, while Lispro and Human Insulin have 30. The B30 amino acid is different among Lispro and Human Insulin as well.
The role of SCFAs on microbiota composition in a mouse model of NTG‐induced migraine
Authors: Lanza Marika , Scuderi Sarah Adriana , Filippone Alessia , Casili Giovanna , Campolo Michela , Paterniti Irene , Cuzzocrea Salvatore , Esposito Emanuela
Published: May 2022

Background

Based on global burden of headache reports, migraine is a prevalent disorder that affect approximately 15% of the adult population. Generally migraine attacks are sporadic, however, some individuals develop a chronic disease form. To date, several researches have shown that migraine is associated with some gastrointestinal disorders such as Helicobacter pylori (HP) infection, irritable bowel syndrome (IBS), and celiac disease (CD). However, the mechanisms explaining how the gut and the brain may interact in patients with migraine are not entirely clear. In this study, we aimed to evaluate the role of the short‐chain fatty acids (SCFAs), such as sodium propionate (SP) and sodium butyrate (SB) as mediators and modulators of host intestinal microbial ecology, in regulating the pathophysiology of migraine in a mouse model induced by nitroglycerine (NTG).

Methods

Mice were orally administered with SB and SP at the dose of 10, 30 and 100 mg/kg, 5 min after NTG intraperitoneal injections. Histological and molecular analysis were performed on the whole brain and small intestine and behavioral tests after 4 h from migraine induction. The composition of the intestinal microbiota was extracted from frozen fecal samples and prepared for sequencing according to the protocol for Illumina Miseq System. However, the expression of inflammatory and oxidative markers were detected by Western blot. Tail flick, hot plate, orofacial formalin and photophobia tests were used to evaluate migraine‐like pain and migraine‐related light sensitivity.

Results

SP and SB treatment notably reduced histological damage in whole brain and small intestine in NTG‐injected mice. Treatments with both SCFAs decreased the markers of inflammation and increased the protective antioxidant enzymes, suggesting an important role of SCFAs to exercise neuromodulatory action. Moreover, SCFAs reduced the headache modifying the intestinal microbiota.

Conclusions

These results provided the evidence that SCFAs exerts a protective effect on central sensitization induced by NTG through a modulation of intestinal microbiota, suggesting a new insight into the potential application of SCFAs as novel supportive therapies for migraine.
Modeling and Analysis of Closed‐Loop Control of the Cardiovascular System
Authors: Gee Michelle , Hornung Eden , Moss Alison , Kuttippurathu Lakshmi , Schwaber James S. , Ogunnaike Babatunde , Vadigepalli Rajanikanth
Published: May 2022

The baroreceptor reflex is a multi‐input, multi‐output physiological control system that regulates short‐term blood pressure by modulating parasympathetic and sympathetic nerve activity between the brainstem and the heart. The opposing effects of parasympathetic and sympathetic nerve activity work in conjunction to maintain cardiovascular homeostasis, with imbalances in activity associated with cardiovascular disease. Recently, attention has focused on the role of the intrinsic cardiac nervous system (ICN) in local control of nervous regulation of the heart and its role in balancing parasympathetic and sympathetic nerve activity. However, it is unknown how the ICN network structure contributes to integrative control of the heart. We formulated multiple alternative network options based on the anatomical, molecular and physiological evidence. We extended a quantitative closed‐loop computational model of the baroreceptor reflex by incorporating a high‐fidelity representation of the ICN to evaluate the impact of altered ICN network structures on overall cardiovascular control. The present computational model consists of (1) a system of ordinary differential equations to represent blood flow in the cardiovascular system, and (2) transfer function representations of sensory neurons, central nervous system neuronal groups, and ICN neuronal groups, connected in a closed‐loop control circuit. We use this model to investigate, via simulation, the role of the intrinsic cardiac nervous system in integrating and modulating parasympathetic and sympathetic nerve activity in healthy and diseased states. Our results show that the local circuit neurons may modulate the ICN network response to distinct vagal inputs towards the integrative control of local cardiac function.
Kidney Injury Stimulates Apical Translocation of NHERF1
Authors: Gagnon Kenneth B. , Bushau‐Sprinkle Adrienne , Hu Ming‐Chang , Chang Audrey N. , Miller R T. , Lederer Eleanor D.
Published: May 2022

Background

NHERF1 (Na+‐hydrogen exchanger regulatory factor isoform 1), recognized as a scaffold protein that regulates proximal tubule phosphate transport, also has a role in regulation of cell metabolism and survival. Our laboratory previously demonstrated more severe cisplatin‐induced acute kidney injury (AKI) in the global NHERF1 knock out (KO) mouse compared to the cisplatin‐treated wild type (WT) mouse. In the present study, we tested the hypothesis that kidney injury results in decreased NHERF1 expression by examining NHERF1 expression in two models of AKI (cisplatin and bilateral renal ischemia‐reperfusion) and two models of chronic kidney disease or CKD (adenine diet and Col4a3‐/‐) by immunofluorescent microscopy and western blot analysis.

Methods

Cisplatin‐induced AKI:Two‐month‐old male and female C57BL/6J WT mice received a single intraperitoneal injection of saline (vehicle) or 20 mg/kg cisplatin. Vehicle‐ and cisplatin‐treated mice were euthanized after 72 hours. Bilateral renal ischemia‐reperfusion injury AKI: Two‐month‐old male and female C57Bl/6J WT mice underwent bilateral renal artery clamp for 30 minutes followed by release. Sham and IRI surgery treated mice were euthanized at 2, 4, and 14 days post‐surgery. Adenine‐induced CKD: Two‐month‐old male and female C57Bl/6J WT mice received either regular or adenine‐supplemented chow for 20 weeks followed by euthanasia. Col4a3‐/‐ CKD: Two‐month‐old male and female C57Bl/6J heterozygous and homozygous KO mice for the collagenase type IV alpha 3 chain were followed for up to 7 months followed by euthanasia. In all four mouse models, the kidneys were removed, de‐capsulated, immersed in 3.7% formaldehyde in phosphate‐buffered saline for 24 hours, then transferred to 70% ethanol for paraffin embedding and immunofluorescent microscopy. Western blots of kidney lysates from each kidney injury model were electrophoresed in 4‐12% Bis‐Tris gradient gels and immunoblotted for NHERF1.

Results

Immunofluorescent microscopy revealed increased staining for NHERF1 at or near the apical membrane of proximal tubule epithelial cells in all four mouse injury models. For the IRI model, longitudinal immunohistochemistry demonstrated a progressive increase in the apical NHERF1 signal from baseline through day 4 post‐surgery followed by a decrease to baseline levels by day 14. Western blot analysis of lysates from all four kidney injury models showed no change in total expression of NHERF1.

Conclusions

We conclude that kidney injury results in translocation of NHERF1 to the apical membrane. These findings suggest that NHERF1 plays a significant role in the response to kidney injury, though at this time, neither the mechanism for translocation nor the specific role of NHERF1 in the response to injury is clear.
N‐methyl‐D‐aspartate Receptor Antagonist Agmatine Prevents Oxycodone Self‐Administration
Authors: Barajas Cecilia , Peterson Cristina , Wilcox George , Fairbanks Carolyn
Published: May 2022

Opioid use disorder (OUD) can be defined as the continued use of opioids despite the negative physical, psychological, and social consequences that result from its use. Since the 1970s, several strategies have been approved by the Food and Drug Administration (FDA) to treat OUD and reverse opioid overdose. Despite their implementation and other exhaustive efforts and resources expended by the scientific and public health communities, the increase in opioid use disorder (OUD) in the United States continues to have staggering economic and quality of life impacts. Decades of research has established that chronic activation of the N‐methyl‐D‐aspartate (NMDA) receptor is a major driving force of the neuroplastic pathology that underlies opioid use disorder. The therapeutic potential of antagonism of the NMDA receptor has been explored, however due to the ubiquity of the receptor, several off‐site side‐effects have hindered development. In recent years, the endogenous NMDA receptor antagonist agmatine has come under consideration for its therapeutic potential in combatting CNS disorders such as neuropathic chronic pain as well as OUD, without producing unwanted side‐effects.

Female ICR mice (21‐30g) were separated into dosing groups and oriented to self‐administration operant chambers in which they were able to press either for oral oxycodone reward (30 mg/L, 200 mg/L, or 1,000 mg/L) or inactive control (H2O + quinine) over 12 hours. Animals were sacrificed and perfused with tissue fixative for later molecular analysis of reward pathways. Animals with the 30 mg/L and 200 mg/L dose option showed consistently higher responding compared to animals pressing for 1,000 mg/L and control. Notably, animals in the 200 mg/L group showed an almost immediate increase in lever presses while the animals in the 30 mg/L group showed a slow increase in responding over time when compared to inactive control. We next sought to characterize agmatine’s ability to prevent oxycodone self‐administration, with 200 mg/L of oral oxycodone selected as the reward dose based on the previous data. Mice were given daily i.p. injections of either agmatine or saline immediately prior to self‐administration sessions. Mice who received agmatine pre‐treatment demonstrated significantly reduced drug‐seeking compared to animals who received saline pre‐treatment. Following completion of the experiment, animals were sacrificed and perfused for molecular analysis.

These data support that agmatine may have therapeutic potential in preventing opioid use disorder. Future studies will focus on comparative molecular analysis between oxycodone‐exposed mice with or without agmatine.
Cortex Specific Overexpression of BAF170 and Growth of the Cranial Base, Cranial Vault, and Facial Skeleton in Mice
Authors: Veltri Megan F. , Durham Emily L. , Lesciotto Kate M. , Coupe Abigail , Kawasaki Mizuho , Motch‐Perrine Susan , Stoykova Anastassia , Tuoc Tran C. , Kawasaki Kazuhiko , Richtsmeier Joan T.
Published: May 2022

The fossil record and craniofacial disease reveal the tight correspondence between brain and skull shape, yet it is not understood how changes in brain growth and consequent changes in brain morphology impact growth of the skull. We tested whether variation in brain size impacted the growth of the cranial base, cranial vault, and facial skeleton in postnatal mice using a transgenic murine model of cortex specific overexpression of Chromatin Remodeling Complex BAF170 Subunit (BAF170cOE) that produces a mouse with a distinctively smaller cerebral cortex. Due to the unique growth mechanisms of the cranial base, cranial vault, and facial skeleton, and their unique anatomic relationships to the cerebral cortex, areas of bone growth may be dissimilarly affected by a reduction in cortical size. Since the cranial vault is anatomically proximate to the cerebral cortex, and because we hypothesized that brain growth impacts skull growth, we anticipated growth of the cranial vault would be more strongly affected relative to growth of the cranial base and facial skeleton. Three dimensional coordinates of landmarks describing the cranial base, cranial vault, and facial skeleton were collected from microcomputed tomography (microCT) images of BAF170cOE mice and unaffected littermates at birth (postnatal day 0 or P0), P14, P28, and P42. Sample sizes ranged between 5 and 10. Growth difference matrix analysis (GDMA) was used to statistically compare growth between genotypes. We found statistically significant differences in cranial vault growth from P0 to P14 between genotypes. There were no significant differences in growth between genotypes for any of the skull modules from P14 to P28 or from P28 to P42. Reduced cortical size of BAF170cOE mice is associated with changes in skull growth during the earliest interval studied here, with significant impacts noted on the growth of the cranial vault. Our results support the hypothesis that brain growth has an effect on skull growth, regardless of the brain decreasing in size. The specificity of the BAF170 Subunit to cause reduced cortical size and lack of direct effect on bone formation allows for future investigation of the mechanisms by which brain and skull growth are integrated.
Single‐Cell Proteomic Profiling of Hepatocytes Reveals Heterogeneous Responses to Hepatotoxic Drugs Nevirapine, Efavirenz, and Acetaminophen
Authors: Yuan Yuting , Orsburn Benjamin , Bumpus Namandjé
Published: May 2022

Drug‐induced hepatotoxicity remains a leading cause of the withdrawal of drugs from the global market. Changes in the proteomic profile of hepatocytes following exposure to known hepatotoxic drugs could provide insight into the mechanisms underlying drug‐induced hepatotoxicity. Thus, we aimed to employ mass spectrometry (MS)‐based proteomics to investigate the impact of a subset of known hepatotoxic drugs, namely, efavirenz (EFV), nevirapine (NVP), and acetaminophen (APAP) on the human hepatocyte proteome in vitro. In addition, to add further granularity to the analysis and to understand potential cell‐to‐cell variability in the proteomic response to the above‐mentioned drugs, single‐cell proteomics was employed.

Cultured cryoplateable hepatocytes were incubated with each drug individually at their IC50 or IC90 values to induce hepatotoxicity. For single‐cell proteomic analysis, after 40‐hour treatment individual hepatocytes were sorted using fluorescence‐activated cell sorting (FACS) and protein lysates were digested with trypsin, labeled with unique tandem mass tags (TMT), and pooled together for multiplex analysis using a timsTOF Flex mass analyzer.

Preliminary single‐cell proteomic results were obtained from 385 cells analyzed at a depth of approximately 350 proteins per cell, and an approximate 1,700 proteins per treatment could be readily quantified using reporter ion‐based quantification with SequestHT with Percolator validation. A principal component analysis (PCA) separated the hepatotoxic drug‐treated cells and DMSO control‐treated cells into two mostly discrete clusters thereby confirming the hepatocytes’ proteome change upon hepatotoxic drug treatment. Further, these data revealed a heterogeneous response to hepatotoxic across single hepatocytes. From proteomic analysis on bulk hepatocytes, we found that the abundance of 36 proteins was significantly increased following exposure of cultured hepatocytes to IC50 and IC90 concentrations of NVP, EFV, and APAP (p‐value < 0.05, Log2 fold change >1). These proteins included calnexin, an endoplasmic reticulum (ER) stress‐induced protein. Interestingly, single‐cell proteomic analysis also showed that the abundance of calnexin was significantly increased following treatment of NVP, EFV, and APAP at IC90; however, the levels of calnexin were divergent across the single cells with certain cells exhibiting no increase in calnexin following drug treatment. These data demonstrate hepatocyte‐to‐hepatocyte heterogeneity in the response to hepatotoxic drugs and suggest that proteomics performed on bulk/total hepatocytes may not fully shed light on hepatoxicity mechanisms.
Ethanol‐Induced Lymphatic Endothelial Cell Permeability: Role of Prox‐1 and Vascular Endothelial Growth Factors
Authors: Hamilton Tiara J. , Molina Patricia , Souza‐Smith Flavia
Published: May 2022

Acute and chronic alcohol (Ethanol, EtOH) administration to rodents promote mesenteric lymphatic vessel permeability leading to immune cell leakage into perilymphatic adipose tissue. We speculate this contributes to impaired immune dialog between the gut and adjacent lymphoid tissue. Mechanistic in vitro studies show that EtOH‐induced lymphatic endothelial cell (LEC) permeability is not associated with alterations in tight and adherens junction expression but is partially rescued by p38 MAP kinase inhibition. Transcription factor Prox‐1 and Vascular endothelial growth factor receptor‐3 (VEGFR‐3) play essential roles in maintaining the integrity of mesenteric lymphatic vessels and of lymphatic vascular functions. The lack of Prox‐1 and VEGFR‐3 expression has been shown to contribute to excessive lymph and vascular leakage, respectively, promoting increased lymphatic permeability. Prox‐1 and VEGFR‐3 are negatively regulated by notch signaling. Notch activation suppresses Prox‐1 and notch target genes, Hey 1 and Hey 2, inhibit VEGFR‐3. We hypothesize that EtOH‐induced LEC permeability is mediated by decreased Prox‐1 and VEGFR‐3 expression via notch signaling activation. We used an in vitro model of rat LEC to characterize Prox‐1 and VEGFR‐3 expression in EtOH‐induced LEC permeability. Cells were seeded in T‐75 flask and cultured with rat endothelial cell medium supplemented with EtOH (0, 25mM, and 50mM) for 30 minutes in an EtOH water bath to limit EtOH evaporation loss from culture media. Gene expression of Prox‐1, VEGFR‐3, Notch 1, Notch 4, Hey 1, and Hey 2 was measured using Quantitative Polymerase Chain Reaction. EtOH at 50mM concentration for 30 minutes significantly decreased gene expression of Prox‐1 and VEGFR‐3 and increased gene expression of Notch 1 and Hey 2. No significant changes were observed in LEC exposed to 25mM EtOH. These findings support our hypothesis that decreases in Prox‐1 and VEGFR‐3 expression involving notch signaling activation may be implicated in EtOH induced LEC permeability leading to mesenteric lymphatic dysfunction.
Impact of Aging on Fiber Cross‐Sectional Areas and Contractile and Fatigue properties of Intrinsic and Extrinsic Tongue Muscles
Authors: Fogarty Matthew , Sieck Gary C.
Published: May 2022

The intrinsic and extrinsic tongue muscles manipulate the position and shape of the tongue and are activated during many oral and respiratory behaviors. In old age, we found that sarcopenia results in selective atrophy of type IIx/IIb diaphragm muscle (DIAm) fibers and a reduction in maximum specific force. We hypothesized that tongue muscles are vulnerable to sarcopenia depending on the relative contribution of type IIx/IIb fibers to total muscle mass. In 6 and 24‐month‐old Fischer 344 rats (n=8 per age), different intrinsic and extrinsic togue muscles were dissected, mounted in a tissue chamber at optimal length and stimulated directly using platinum plate electrodes. Maximum isometric force and fatigue properties were then determined. After mechanical measurements, muscle segments were frozen at optimal sarcomere length in melting isopentane. Transverse sections of muscle fibers were cut at 10 µm, and fiber type was determined based on immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. Muscle fiber images were acquired using a confocal microscope (40x 1.3NA objective), and fiber cross‐sectional areas as well as interstitial space were measured. In superior and inferior longitudinal intrinsic muscles specific force was reduced by ~25% in older rats and type IIx/IIb fibers were ~20% smaller. In the genioglossus muscle, specific force was reduced by ~20% in older rats, and the cross‐sectional areas of type IIx/IIb fibers were ~20% smaller. There were no age‐related changes in specific force or fiber cross‐sectional areas in the transversalis muscle. The residual force following 2‐min of fatiguing contractions remained similar in young compared to old rats, consistent with the selective effect of sarcopenia on type IIx/b fibers. The results of this study support our hypothesis that tongue muscles are susceptible to age‐related sarcopenia. The presence of sarcopenia may contribute to the increased incidence of obstructive sleep apnea in older individuals.