bluuebunny/crossref_metadata_2025 huggingface dataset:
Catestatin regulates core bioenergetic and metabolic functions of the myocardium
Authors: Mahata Sushil K. , Mahata Sumana , Pasqua Teresa , Avolio Ennio , Tang Kechun , Bandyopadhyay Gautam , Webster Nicholas J. G.
Published: May 2022

Objective

Hypertension is a global pandemic, affecting more than one billion people. We have recently reported that chromogranin A (CgA) peptide catestatin (CST: human CgA352‐372) decreases blood pressure in hypertensive CST knockout (CST‐KO) mice by inhibiting catecholamine secretion and decreasing inflammation. Using CST‐KO mice we show here that CST regulates core bioenergetic and metabolic functions of the myocardium.

Methods and Results

We carried out transcriptomic analyses (gene microarray) of the left ventricles of wild‐type (WT) and CST‐KO mice. Analysis revealed altered expression. Downregulated genes, which were most enriched in pathways that impact muscle conduction and contractility include, among other genes, Glo1 (glyoxylase 1). Upregulated genes include, among others, Gm7120 (predicted gene 7120), Astn2 (astrotactin 2), and Defa20 (defensin a‐20). We validated several of these up‐ and downregulated genes by qPCR and found that CST supplementation reversed these aberrant expressions in all cases. Targeted qPCR analysis of sarcomeric genes revealed reduced expression of Tnni3 (cardiac troponin I), Tnnt2 (cardiac troponin T), Myl4(atrial light chain‐1) and Myl7 (atrial light chain‐2) genes in CST‐KO mice. CST treatment increased expression of Tnni3, Tnnt2, Actc1 (cardiac a‐actin), Myl4 and Myl7 in both WT and CST‐KO mice albeit to a much higher level in CST‐KO mice. An analysis of mitochondrial genes revealed reduced expression of Ndufa3 (complex I), Sdhc (complex II) and Atp5j (complex V) genes in CST‐KO mice. CST treatment increased the expression of all three genes in WT and CST‐KO mice. Taken together, these unbiased approaches and qPCR validation studies provide insight into how an inflamed dysfunctional heart in the absence of CST may impact muscle function (contractility), rhythm, mitochondrial bioenergetics, and conduction.

CST‐KO mice showed decreased uptake of 2‐deoxy glucose in the left ventricle compared to WT mice. Furthermore, glucose‐6‐phosphate utilization was significantly lower in CST‐KO compared to WT mice. We also found that compared to WT mice, CST‐KO mice show a significantly higher uptake of fatty acids. Furthermore, 14C‐labeled acid soluble metabolites were significantly higher in CST‐KO compared to WT mice, suggesting that increased dependence on fatty acids for energy needs make CST‐KO hearts resistant to insulin.

Conclusions

Taken together with the cardiometabolic shifts (decreased glucose utilization with a concomitant increase in the utilization of fatty acids), these findings indicate that CST maintains cardiometabolic homeostasis; it does so at least in part by acting as an insulin‐sensitizing peptide which regulates myocardial choice of substrates for generating energy (i.e., ATP).
Chromogranin A plays a crucial role in age‐related development of insulin resistance and hypertension
Authors: Mahata Sushil K. , Liu Matthew A. , Jati Suborno , Tang Kechun , Bogaart Geert , Ghosh Gourisankar
Published: May 2022

Objective

Aging is a public health, societal, and economic problem around the world. Adults aged 65 years and older are the fastest growing segment of the US population, and their number is expected to double to 89 million by 2050. Two physiological processes that are more prevalent in older populations are hypertension and diabetes. Chromogranin A (protein: CgA; mouse gene: Chga) knockout (Chga‐KO) mice are associated with hypertension but also protected against developing autoimmune diabetes; however, the effect of these phenotypes on mice as they age including mortality remains unclear, as does a possible mechanism for developing both phenotypes. This study examined the long‐term effects of aging in Chga‐KO mice and sought to elucidate a possible mechanism for both hypertension and diabetes by focusing on the inflammatory pathway.

Methods and Results

Chga‐KO mice display two opposite aging phenotypes: hypertension but heightened insulin sensitivity. We determined these phenotypes in aging CgA mice by measuring blood pressure and blood glucose. In comparison, aging wild‐type (WT) mice gradually lost glucose tolerance and insulin sensitivity. Moreover, while aging WT mice had increased inflammation with higher plasma TNF‐a, IFN‐g and CCL2 and increased mitochondrial fission, these phenotypes were the opposite in aging Chga‐KO mice. Chga‐KO mice also showed increased expression of mitochondrial and nuclear‐encoded complex I genes, implying that they were healthier than WT mice. Most intriguingly, the hypertension in Chga‐KO mice spontaneously reversed with aging. Supplementation of Chga‐KO mice with pancreastatin (human CgA273‐301), a hyperglycemic peptide produced from CgA by proteolysis, increased both blood glucose levels and blood pressure, implicating hyperglycemia, and hypertension.

Conclusions

We conclude that age‐related insulin resistance and hypertension are caused in part by CgA.
Trichinosis of Marine Mammals in the Territory of Chukotka
Authors: Mashkina Darja , Bukina Lidiya , Gaponova Viktoriya
Published: May 2022

Chukotka is the only region of the Russian Federation where traditional fishing of marine mammals, including the gray whale Eschrichtius gibbosus, is conducted. More than 70% of the indigenous population of the coastal settlements of the Chukchi Peninsula eat the meat of marine mammals. Due to the huge distances between communities, lack of infrastructure, limited access to medical and veterinary care, isolated studies of marine mammal infestation with trichinella larvae are being conducted.

For the first time, trichinella in the Pacific walrus Odobenus rosmarus divergens in Chukotka were recorded in the coastal zone of the Bering and Chukchi Seas in the 1970s. In the future, trichinosis has not been studied in this region, which confirms the relevance of our work. Infection of marine mammals was studied by trichinelloscopy (compressor and biochemical). Bioassays were collected during fishing by sea St. John's wort in the village of Lorino. Infection rates were determined. Studies conducted on the Chukchi Peninsula confirmed the infection with trichinosis of the Pacific walrus and revealed the larvae of trichinella in lakhtak Erignathus b.nauticus and ringed seal Phoca hispida, and later in larga Phoca largha. The extent of invasion varied from 0.9% in larga to 4.3% in lakhtak. The gray whale is the most popular food item in the diet of the indigenous inhabitants of Chukotka. According to the results of the research, out of 119 individuals of gray whales, trichinella were not found. Given the trophic‐chorological connections of marine mammals, it is difficult to imagine a way of transmission of trichinella in marine ecosystems. However, previously obtained research results allow us to do this. On the Arctic coasts, especially near settlements, there are a large number of corpses of dogs, cats, rats, voles, carcasses of small seals that fall into marine ecosystems. Invertebrates, their adult and larval forms, as well as vertebrates ‐ fish and birds ‐ occupy an important place in the process of disposal of corpses. Studies have shown that various types of invertebrates from 1 to 59 hours, and vertebrates from 28 to 95 hours can be direct or indirect sources of the pathogen of invasion and contribute to their mechanical transfer. Thus, in the conditions of coastal Arctic marine ecosystems, successful circulation of trichinella is carried out, thanks to human economic activity and the specifics of the trophic‐horological relationships of obligate and potential host species. Therefore, monitoring of trichinella invasion in these regions is vital.
Upregulation of Hippocampal MAPKERK/NFκB Signaling Accounts for the Opioid Receptor‐dependent Incitement of Cognitive Impairment in Septic Rats
Authors: Abuiessa Salwa A. , Ayieng’a Evans O. , Elblehi Samar S. , El‐Gowilly Sahar M. , Afify Elham A. , El‐Mas Mahmoud M.
Published: May 2022

Earlier reports indicate that morphine provokes cognitive and circulatory defects caused by sepsis. Pharmacologic, histopathologic, and molecular studies were employed in this communication to implicate inflammatory pathways of the hippocampus in this clinically relevant sepsis‐morphine interaction. Sepsis was induced with cecal ligation and puncture and behavioral studies were undertaken 24 hr later. The data showed that the subcutaneous treatment of septic rats with morphine (7 mg/kg, 6 and 24 hr prior to sepsis) caused significant (i) decreases in systolic blood pressure measured by the tail‐cuff plethysmography, and (ii) impairment in cognitive function as revealed by Morris water maze test, new object recognition test, and Y maze test. Such hemodynamic and behavioral anomalies were substantially obliterated after blockade of opioid receptors or inhibition of TNFα with naloxone (0.5 mg/kg) and infliximab (6 mg/kg), respectively. The improvement in cognitive function caused by naloxone or infliximab was paralleled with dramatic falls in serum IL‐1β and diminution in neuronal degeneration and necrosis seen in hippocampal tissues. Immunohistochemical studies revealed that the heightened hippocampal expression of MAPKERK and NFκB observed in the sepsis/morphine‐challenged rats was remarkably reduced upon treatment with naloxone or infliximab. These findings incriminate opioid receptors in the amplified hemodynamic and behavioral sequalae of sepsis and suggest a therapeutic potential for the downregulation of hippocampal MAPKERK/NFκB cascade in offsetting the exaggerated responsiveness to the sepsis/morphine insult.
A Truncated Mutant – The Most Common Sucrase‐Isomaltase Deficiency in the Inuit Population: Cellular and Biochemical Characterization
Authors: Tannous Stephanie , Naim Hassan Y.
Published: May 2022

Sucrase isomaltase (SI) is the most prominent disaccharidase in the small intestine responsible for the final steps of carbohydrate digestion. Mutations in the SI gene can lead to a drastic reduction or loss of the catalytic activity required for digestion of disaccharides, and thus can be associated with maldigestion and malabsorption of carbohydrates. Carbohydrate malabsorption is one of the most common gastrointestinal problems, where 70% of the population are affected. Congenital sucrase‐isomaltase deficiency (CSID) is an autosomal recessive disorder caused by defective SI activity; and it is clinically characterized by abdominal pain, flatulence, bloating, and diarrhea. Recently, a homozygous frameshift mutation, c.273_274delAG (p.Gly92Leufs*8), was identified in the Inuit population with an observed allele frequency of 17.2%. In this study, Cos‐1 cells were transiently transfected with cDNA encoding the Gly92Leufs*8 mutation or SI wild‐type (SI‐WT) as a control. The assessment of this new variant phenotype was investigated through enzymatic activity measurements, cellular localization and trafficking behavior. The truncation is associated with the elimination of all N‐glycosylation sites, whereas the Ser/Thr‐rich stalk region is retained pointing to a potential O‐glycosylation. In fact, treatment with benzyl 2‐acetamido‐2‐deoxy‐α‐D‐galactopyranoside, an inhibitor of O‐glycosylation in the Golgi, resulted in a substantial shift in the size of the mutant concomitant with transport competence of the mutant to O‐glycosylation in the Golgi Apparatus. The mutant is further trafficked to the cell surface as assessed by the detection of biotinylated forms of the mutant at the cell surface. This result was also corroborated by immunofluorescence images, which revealed the mutant at the cell surface. However, this mutant does not exhibit SI activity due to the absence of the sucrase and isomaltase domains. While our data unraveled the molecular basis for the onset of the clinical symptoms in patients homozygous to this mutation, further work is needed to determine whether heterozygotes with this mutation are also affected and to which extent.
Developing therapeutic protein disaggregases for Neurodegenerative Disease
Authors: Lin JiaBei , Shorter James
Published: May 2022

The accumulation of abnormal protein aggregates in human brain is connected with neurodegenerative diseases. The mammalian protein‐disaggregase system comprised of Hsp110, Hsp70, and Hsp40 can protect cells from proteinopathies. However, this disaggregase system has limitations and its activity declines with age. As a therapeutic strategy for neurodegenerative disease we propose to introduce an exogenous, synthetic disaggregase machinery, based on Hsp104 that converts amyloid to soluble, functional protein. Hsp104 is a hexameric AAA+ ATPase that is critical for stress tolerance in yeast by facilitating the resolubilization of stress‐damaged, aggregated proteins. The homologues of Hsp104 are highly conserved in bacteria, fungi, and plants, but absent from metazoa. The disaggregase activity of Hsp104 can be enhanced by the presence Hsp70 and Hsp40. Hsp104 can dissolve α‐synuclein, β‐amyloid, and TDP‐43 aggregates, but impractically high Hsp104 concentrations are needed. We aim to rationally design potentiated Hsp104 variants that can reverse protein misfolding at low concentrations. One potential problem for the currently discovered potentiated Hsp104 variants is their “off‐target” effects, where expression potentiated Hsp104 variant in ∆hsp104 yeast causes cell death possibly by unfolding metastable proteins at 37°C. By investigating the mechanism of Hsp104 disaggregation and its structural basis, we will engineer and evolve potentiated Hsp104 variants with minimized “off‐target” effect that eradicate α‐synuclein, β‐amyloid, or TDP‐43 misfolding and toxicity. We have exploited new cryo‐EM structures of Hsp104 to engineer key interfaces to yield several novel Hsp104 variants with tunable activity, which can reverse protein aggregation in the presence or absence of human Hsp70 and Hsp40. These novel Hsp104 variants effectively rescue the aggregation and toxicity of diverse human neurodegenerative disease proteins and could have therapeutic utility. Moreover, our engineered Hsp104 variants provide key mechanistic insights into disaggregase function.
Intracellular Sphingosine‐1‐Phosphate is Involved in Lung Cancer Cells Proliferation
Authors: Terlizzi Michela , Colarusso Chiara , Pinto Aldo , Sorrentino Rosalinda
Published: May 2022

Background

Sphingosine‐1‐phosphate (S1P) is a membrane‐derived bioactive phospholipid exerting a multitude of effects on respiratory cell physiology and pathology through five S1P receptors (S1PR1‐5). Higher levels of S1P have been registered in a broad range of respiratory diseases, including inflammatory disorders and cancer, although its exact role is still elusive.

Objectives

The aim of our study was to understand the role of S1P in healthy versus lung adenocarcinoma cells after Toll‐Like Receptors (TLRs) activation, well‐known modulators of sphingolipid metabolism.

Methods

We stimulated healthy fibroblast and lung adenocarcinoma cells with TLRs ligands and S1P, to compare physiological vs pathological S1P role. Pro‐inflammatory cytokines release, proliferation rate and the expression/activity of the mediators involved in the S1P signaling were evaluated.

Results

We found that the activation of TLR9, but not of TLR4, was involved in the generation of S1P by means of ceramidase and sphingosine kinase I/II (SPHK I/II) activation in tumor cells but not in non‐cancerous cells. Through an inside‐out signaling, newly formed S1P was extruded from the cells through its transporter SPNS2, supporting TLR9/NF‐kB‐mediated TNF‐α release mainly via membrane S1PR3. These results were not observed in healthy fibroblasts, implying that this pathway was mainly involved in pathological conditions. Moreover, we found that S1PR3 and SPHK II were overexpressed in the nucleus of lung adenocarcinoma cells and that were associated to S1P‐mediated proliferation of tumor cells.

Conclusion

These results prove that the endogenous TLR9‐induced S1P can on one side favor pro‐inflammatory mechanisms in the tumor microenvironment via the activation of cell surface receptors, but on the other tumor progression via the nuclear S1PR3/SPHK II axis, highlighting a novel molecular mechanism that identifies S1P as one of the crucial mediators for lung carcinogenesis‐associated inflammatory processes.
PAR2, not PAR1, regulates endothelium‐dependent vascular tone in resistance arteries
Authors: Zhang Xun , Lee Matthew D. , Buckley Charlotte , Wilson Calum , Hollenberg Morley , McCarron John G.
Published: May 2022

Protease activated receptors (PARs), a subfamily of G‐protein‐coupled receptors, are activated by cleavage of its extracellular domains. Four mammalian PAR members have been identified (PAR1‐4). PAR1 is the thrombin receptor, while PAR2 is activated physiologically by peptides or N‐terminal proteolytic cleavage. The resulting cleaved N‐termini acts as a tethered ligand that initiates receptor signaling. PARs may regulate vascular tone, cell permeability and cell proliferation. However, the precise roles of PAR1 and PAR2 in regulating vascular tone via the endothelium, and how PAR‐evoked signals transmit extracellularly and intracellularly, are incompletely understood.

To explore the physiological functions, and the underlying signalling pathways in native endothelium, PAR1 (TFLLR‐NH2) and PAR2 (2‐Furoyl‐LIGRLO‐amide; 2fLI) activating peptides were used, and changes in intracellular Ca2+ and contractility measured in en face rat mesenteric arteries. Surprisingly, PAR1 activation (TFLLR‐NH2or trypsin) neither triggered Ca2+ signals in endothelial cells or alter vascular tone. On the other hand, PAR2 activation (2fLI) evoked large global propagating Ca2+ waves in endothelial cells and endothelium‐dependent dilation of mesenteric arteries. The Ca2+ signals evoked by PAR2 activation were kinetically distinctive when compared to those of muscarinic receptor activation. PAR2 activation (2fLI) also evoked responses in different clusters of endothelial cells from those of muscarinic receptor (acetylcholine) activation. PAR2 activation with 2fLI or trypsin failed to evoke Ca2+ signals after depletion of the internal store with the Ca2+‐ATPase (SERCA) inhibitor, CPA. This result suggests that PAR2 evokes Ca2+ release from internal stores. Pharmacological block of inositol 1,4,5‐trisphosphate (IP3) with either 2‐APB or caffeine, also blocked PAR2‐evoked Ca2+ signals. Phospholipase C inhibition (U73122) also blocked the Ca2+ signals evoked by PAR2 activation. Therefore, PAR2 is a PLC linked G protein coupled receptor which evokes Ca2+ release from the internal store via IP3 receptors in endothelial cells. The TRPC3 blocker, PYR3 and store‐operated Ca2+ entry (SOCE) blocker, YM58483 each significantly supressed PAR2 mediated Ca2+ signals, suggesting internal store was replenished via TRPC3 channel and SOCE.

In conclusion, PAR2 rather than PAR1, regulates vascular tone in mesenteric arteries by evoking Ca2+ release from the internal store via IP3 receptors to generate propagating Ca2+ waves in distinct sub‐populations of endothelial cells.
Ruxolitinib Prevents Ventilator Induced Diaphragm Dysfunction
Authors: Addinsall Alex B. , Cacciani Nicola , Akkad Hasem , Moruzzi Noah , Maestri Alice , Shevchenko Ganna , Bergquist Jonas , Ruas Jorge , Larsson Lars
Published: May 2022

Mechanical ventilation (MV), however brief results in the loss of diaphragm muscle mass and strength, termed ventilator induced diaphragm dysfunction (VIDD). VIDD increases dependence, complicates and prolongs weaning and significantly increases discharge mortality rate and health care costs worldwide. The Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway was recently identified as an important signalling pathway implicated in VIDD, upregulated in the diaphragm following MV and limb muslces during critical care. Regulation of STAT3 is imperritve to skeletal muscle mass and function, as STAT3 is required in proper muscle growth and regeneration, while chronic activation of STAT3 is implicated in muscle dysfunction. As JAK/STAT pathway inhibition can restrict the development of chronic muscle wasting conditons, this study aimed to explore the therapeutic potential of Ruxolitinib, an approved JAK1/2 inhibitor for myelofibrosis, for treatment of CIM. We hypothesised Ruxolitinib would reduce loss of muscle mass and function associated with VIDD. Here, rats were subjected to five days controlled MV (CMV) with and without daily Ruxolitinib gavage. Five‐days CMV significantly reduced diaphragm muscle size and impaired specific force, which was associated with 2‐fold upregulation of P‐STAT3, disrupted mitochondrial structure and respiratory function. Expression of the motor protein myosin was not affected, however CMV may alter myosin function through deamidation post translational modification. Ruxolitinib increases five‐day survival rate, restored P‐STAT3 expression and preserved diaphragm muscle size and specific force. These functional improvements were associated with improved mitochondrial structure, augmented mitochondrial respiratory function and reversal or augmentation of myosin deamidations. These results provide evidence of the preclinical potential of repurposing Ruxolitinib for the treatment of VIDD.
Transcriptional response of aldosterone target genes in the rat and human distal colon during hepatic cirrhosis
Authors: de la Rosa Diego Alvarez , Serrano‐Morillas Natalia , González‐Alayón Carlos , Shehwana Huma , Konu Ozlen , Hernández‐Guerra Manuel
Published: May 2022

Hepatic cirrhosis is associated to circulatory abnormalities. A combination of vasodilation, lower plasmatic protein content and splanchnic venous stagnation leads to hypovolemia, which in turn stimulates the renin‐angiotensin‐aldosterone system (RAAS). Advanced stages of the disease cause renal failure and subsequent impaired K+ homeostasis. It has been proposed that the distal colon undergoes functional remodeling during renal failure, in particular by aldosterone‐driven increased K+ excretion. In this study, we compared the transcriptional response of aldosterone target genes in the rat distal colon under two models of increased circulating aldosterone, one with concomitant RAAS activation, and in a model of hepatic cirrhosis. We examined known aldosterone‐regulated transcripts involved in corticosteroid signaling and transepithelial ion transport. In addition, we included new aldosterone‐regulated genes identified via whole transcriptome analysis. Our comparison revealed that multiple aldosterone‐target genes are upregulated during cirrhosis in the rat distal colon. This upregulation is more prominent in animals showing decompensated cirrhosis. Epithelial Na+channel (ENaC) β and γ subunit expression correlated positively with plasma aldosterone concentration and negatively with glomerular filtration rate. Altogether, our results demonstrate cirrhosis‐induced ion transporter subunit expression remodeling, a change that correlated well with the severity of the disease and suggested a role for aldosterone in the process. The expression of a subset of these genes was then tested in distal colon biopsies obtained from patients showing decompensated cirrhosis and treated or not with mineralocorticoid receptor inhibitor spironolactone. Preliminary results show decreased expression of ENaC subunits and channel regulator K‐ras in patients treated with spironolactone. We conclude that cirrhosis progression towards a decompensated state with hypovolemia induces remodeling of distal colon ion transporter expression to match a decaying kidney function.
A Budding Yeast Model System to Define Biological Pathways Altered by Pathogenic Missense Mutations in Histone Genes Identifies a Link between Histone H3K36 and the TOS4 Gene
Authors: Ambrocio Reina E. , Lemon Laramie D. , Kannan Sneha , Mo Kim Wai , Adams Miranda , Fasken Milo B. , Spangle Jennifer M. , Corbett Anita H.
Published: May 2022

Histones are critical for both packaging DNA into chromatin within the cell nucleus and for regulating all aspects of gene expression. DNA is packaged into nucleosomes, which consist of a hetero‐octamer of histones H2A, H2B, H3, and H4. Somatic missense mutations in histone genes turn these essential proteins into oncohistones, which can drive oncogenesis. These mutations, termed oncohistone mutations, have been linked to multiple types of cancer, including pediatric gliomas and head and neck cancers. In humans, a histone H3 lysine 36 to methionine missense mutation (H3K36M) causes chondroblastomas. Our lab has employed a budding yeast model system to explore how missense mutations such as H3K36M alter histone function. These studies, as well as those from previous groups, reveal that changes at H3K36 confer sensitivity to growth on plates containing caffeine, suggesting that H3K36 mutants show changes in stress response pathways. To further define these pathways, the laboratory employed a high copy suppressor screen to identify genes that when overexpressed could suppress the caffeine‐sensitive growth phenotype of H3K3M cells. This screen identified several genes linked to epigenetic regulation, including TOS4, which encodes a forkhead‐associated (FHA) domain protein that interacts with Rpd3L and Set3 histone deacetylase complexes. To begin to define the function of Tos4 required for this suppression, two amino acid substitutions within the Tos4 FHA domain that disrupt interaction with histone deacetylase complexes (R122A, N161A) were generated. This Tos4 variant still suppresses the caffeine‐sensitive growth of H3K36M cells, suggesting these interactions are not required for suppression. My studies focus on defining the function of Tos4 required for this suppression. Results from such studies may provide insight for novel therapeutic targets in humans diagnosed with these distinct oncohistone‐driven cancer types.
Validation of a new assessment tool for chest tube insertion on Thiel embalmed human cadavers: a feasibility study
Authors: De Mol Leander , Willaert Wouter
Published: May 2022

Intro

Thiel embalmed human cadavers are considered as high‐fidelity simulator for several surgical procedures, including Chest Tube Insertion (CTI). In proficiency‐based education, targeted feedback is essential in order to improve learners’ technical skills. Assessments must be validated prior to their use. Validity can be established using Messick’s framework, which collects evidence from five sources. The ACTION tool is a new assessment tool, and consists of a procedure‐specific Rating Scale and an error checklist. It has been developed by a Delphi panel, which ensures content evidence. This study evaluates the feasibility of a validation study for the ACTION tool on Thiel embalmed human cadavers.

Methods

A convenience sample of three medical undergraduates and one PhD candidate was recruited. All provided informed consent and demographic information. Each participant was scored and recorded while performing a CTI. Each recording was later scored by three raters. Rater agreement was evaluated by calculating a two‐way mixed effects, absolute agreement, single unit of measurement intraclass correlation coefficient (ICC) model. Internal consistency of the assessment was evaluated using Cronbach Alfa. Feasibility was evaluated using structured questions regarding recruitment and data outcomes.

Results

Recruitment capability and data collection procedures were evaluated as being feasible and suitable for the target study participants. All necessary resources are available and data analysis can be carried out.

Preliminary results for the procedural steps indicate an ICC of 0.62, indicating moderate rater agreement, and a Cronbach Alfa of 0.94, indicating excellent internal consistency. For the errors, an ICC of 0.63, and a Cronbach Alfa of 0.68 was observed.

Conclusion

Validation of the ACTION tool following Messick’s framework is feasible. A rater training session can increase reliability. After validation, the assessment tool can be used to evaluate learners’ proficiency in CTI on Thiel embalmed human cadavers, prior to patient contact.
Gentamicin induces calciuresis by blocking TRPV5
Authors: van Megen Wouter H. , Beggs Megan R. , An Sung‐Wan , Ferreira Patrícia G. , Lee Justin J. , Wolf Matthias T. , Alexander R. T. , Dimke Henrik
Published: May 2022

Background

Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe side effects including renal Ca2+wasting. The underlying mechanism is unknown but proposed to involve activation of the Ca2+‐sensing receptor (CaSR) in the thick ascending limb (TAL), which would increase expression of claudin‐14 (CLDN14) and limit Ca2+ reabsorption. However, no direct evidence for this hypothesis has been presented.

Methods

The effect of gentamicin on renal Ca2+ handling was studied in vivo using mouse models with impaired Ca2+ reabsorption in the proximal tubule and TAL. The effects of gentamicin in CaSR activation were studied in vitro using a Cldn14 promoter luciferase‐reporter assay, while the effect of gentamicin on the distal nephron Ca2+ channel transient potential receptor vanilloid 5 (TPRV5) activity was determined by patch‐clamp in HEK293 cells.

Results

Gentamicin increased urinary Ca2+ excretion in wildtype mice following acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and remained present in furosemide‐treated animals. Moreover, gentamicin‐induced calciuresis was not significantly different in Cldn14‐/‐ mice or mice with impaired proximal tubular Ca2+reabsorption (Cldn2‐/‐ mice), compared to wildtype animals. In vitro, gentamicin failed to activate the CaSR. In contrast, patch‐clamp analysis revealed that gentamicin inhibited rabbit and human TRPV5 channel currents in a dose‐dependent manner. In accordance with a direct effect of gentamicin on TRPV5, chronic gentamicin administration downregulated gene expression of distal nephron Ca2+ transporters in mice.

Conclusion

We show here that gentamicin does not cause hypercalciuria via activation of the CaSR‐CLDN14 pathway or by interfering with proximal tubular CLDN2‐dependent Ca2+ reabsorption. Instead, gentamicin blocks distal Ca2+ reabsorption by direct inhibition of the Ca2+ channel TRPV5. These findings offer new insights into Ca2+ wasting in patients treated with gentamicin.
Electronic Nicotine Delivery Systems Exhibit LowerToxicity Compared to Cigarettes: “The Replica Study Experience”
Authors: Distefano Alfio , Caruso Massimo , Emma Rosalia , Rust Sonja , Poulas Konstantinos , Zadjali Fahad , Boffo Silvia , Volarevic Vladislav , Mesiakaris Konstantinos , Karanasios Georgios , Al Tobi Mohammed , Albalushi Najwa , Giordano Antonio , Canciello Angelo , Arsenijevic Aleksandar , Ilic Aleksandar , Caruso Tancredi , Carota Giuseppe , Spampinato Maria R. , Zuccarello Pietro , Ferrante Margherita , Polosa Riccardo , Li Volti Giovanni
Published: May 2022

Electronic nicotine delivery systems (ENDS) can reduce the health risks associated with chronic smoke exposure, and their potential benefits are the subject of intense scientific debate. The international “Replica Study Group” replicated independently three relevant studies from the tobacco industry on the cytotoxic and inflammatory effects of cigarette smoke and ENDS aerosol. Our primary goal was to establish the reliability of the results and the robustness of the conclusions.

In order to assess cytotoxicity of smoke and aerosol, we exposed human bronchial epithelial cell (H292) to cigarette smoke and to ENDS aerosol at air‐liquid interface (ALI). Moreover, we aimed to assess different inflammatory and remodeling mediators release (IL‐6, IL‐8 and MMP‐1) from cells exposed to whole smoke (WS) and to smoke deprived of total particulate matter (vapor phase; VP).

We were able to replicate the results obtained in the original studies on cytotoxicity confirming that almost 80% of the cytotoxic effect of smoke is due to the vapor phase of smoke. Moreover, our results substantiated the significantly reduced cytotoxic effects of ENDS aerosol vs. cigarette smoke. However, our data were notably different in terms of inflammatory and remodeling activity triggered by smoke.

Altogether, the data obtained independently in different laboratories clearly confirm the reduced toxicity of ENDS products compared to smoke, thus providing a valuable tool to the harm reduction strategies in smokers. Otherwise, we were not able to replicate the results on inflammatory and remodeling mediators released from smoke exposed cells. This could be due to the lack of normalization of the quantity of cytokines released, with the number of viable cells, in the original paper, since the production of cytokines itself requires the metabolic capability of cells.
5‐HT7 Receptors Mediate Dilation of Rat Cremaster Muscle Arterioles in vivo
Authors: Jackson William F. , Daci Armond , Thompson Janice , Fink Gregory D. , Watts Stephanie W.
Published: May 2022

Serotonin (5‐HT) infusion in vivo causes hypotension and a fall in total peripheral resistance (TPR) that is mediated by 5‐HT7 receptors. 5‐HT dilates small arterioles in skeletal muscle, a microcirculation that importantly contributes to TPR. However, the receptors that mediate this dilation have not been established. Therefore, we tested the hypothesis that 5‐HT7 receptors mediate the arteriolar dilation in the skeletal muscle microcirculation. Cremaster muscles of isoflurane‐anesthetized male Sprague‐Dawley rats were prepared for digital in vivo microscopy and superfused with physiological salt solution (PSS) at 34 °C. Bolus (1‐10 nmols) application of 5‐HT into the superfusate caused substantial dilation of 3rd to 5th order arterioles from 10 ± 1 to 27 ± 1 μm (n = 10 arterioles from 4 rats, p <0.0001) that recovered within 2‐3 min. Dilation to bolus application of 5‐HT was inhibited by superfusion with 1 μM SB269970, a selective 5‐HT7 receptor antagonist (diameter in SB = 9 ± 1 μm; diameter in SB + 5‐HT = 10 ± 1 μm; n = 10 arterioles from 4 rats; p = 0.8763). Similarly, steady‐state superfusion with 10‐30 nM of the 5‐HT1/7 receptor agonist 5‐carboxamidotryptamine (5‐CT) dilated 3rd‐5th‐order arterioles from 16 ± 3 to 37 ± 5 μm (n = 7 arterioles from 3 rats, p = 0.0004). This dilation was reversed (diameter in 1 μM SB269970 = 13 ± 2 μm, n = 7, p = 0.1479 vs. baseline) or blocked (diameter in 1 μM SB269970 + 10‐30 nM 5‐CT = 10 ± 2 μm, n = 7, p = 0.0005 vs. 5‐CT alone) by 1 μM SB269970. Dilation of arterioles to the muscarinic agonist, methacholine (MCH) was not inhibited by SB269970 verifying the specificity of the antagonist; arterioles dilated from 13 ± 2 μm to 25 ± 2 μm to 100 nmols MCH and dilated from 9 ± 2 μm to 32 ± 2 μm in response to MCH in the presence of 1 μM SB269970; n = 10 arterioles from 4 rats; p = 0.7232 vs. dilation in the absence of SB269970. Consistent with these data, pooled samples (4‐5 rats) of 1st and 2nd – order cremaster arterioles expressed message for 5‐HT7 receptors by quantitative real‐time PCR (CT = 36.4 ± 0.74, n = 3 pooled samples, p = 0.039 vs CT = 40). These data support our hypothesis that 5‐HT7 receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to the observed hypotension when these receptors are engaged in vivo.
Hypoxia Decreases Jump Rate and Increases Lactate Production in Late‐Stage Grasshoppers
Authors: Heiting Grace M. , Vanasse Sarah B. , Kirkton Scott D.
Published: May 2022

Insects are the most evolutionarily successful animals in part due to their air‐filled tracheal respiratory system. The tracheal system is an intricate network of tubes that exchange gases between the atmosphere and tissues. Some insects, including grasshoppers, also have air sacs that enhance convective airflow. During intermolt periods, the American locust (Schistocerca americana) doubles their body mass, which may compress the tracheae. In a recent study, late‐stage grasshoppers had reduced air sac ventilation frequency, decreased convective capabilities, and less of their body dedicated to their respiratory system. We hypothesized that these developmental changes may reduce both oxygen delivery and jump performance. We forced early‐stage (day 2) and late‐stage (day 8) 6th instar grasshoppers to jump in artificial oxygen atmospheres (5%, 10%, 21%, 40% oxygen: balance nitrogen) in a 100 L gloved box. After jumping, grasshoppers were immediately frozen in liquid nitrogen for a fluorometric lactate assay. We predicted that if oxygen delivery is compromised during development, then late‐stage grasshopper jump rate and lactate production would correlate with oxygen level.

Within the first one to two minutes of jumping, there were no significant differences in jump rate between early‐ and late‐stage grasshoppers at any oxygen level. This result was not surprising since previous work has shown that the first two minutes of jumping is fueled primarily by anaerobic ATP production. At moderate hypoxia (10% O2) and normoxia (21% O2), there were no differences in jump rate between early‐ and late‐stage grasshoppers. In extreme hypoxia (5% O2), there was no difference in jump performance for the first two minutes, but late‐stage grasshoppers had a 1.6‐fold lower average jump rate in minutes three through five. In hyperoxia (40% O2), jump performance was similar during the first minute, and late‐instar grasshoppers jump rates increased on average by 1.4‐fold in minutes two through five.

Late‐instar grasshopper muscles produced significantly greater lactate regardless of oxygen level. Within each group, oxygen level did not significantly affect jumping muscle lactate production. However, late‐stage grasshoppers produced 26.3% significantly more lactate in 5% oxygen than 40% oxygen (post‐hoc Student’s t; p<0.04). Future research will examine how age and oxygen level impact HIF‐1 expression in grasshoppers. Since late‐stage grasshoppers have lower jump rates and increased lactate production, we predict they will also have elevated HIF‐1 protein levels.
Targeting Diabetic Cardiomyopathy: LncRNA Kcnq1ot1 Rescues Mitochondrial ATP Synthase via Sponging of MiR‐378a‐5p
Authors: Durr Andrya J. , Hathaway Quincy A. , Kunovac Amina , Taylor Andrew D. , Rizwan Saira , Cook Chris C. , Hollander John M.
Published: May 2022

Diabetes mellitus has been linked to an increase in mitochondrial microRNA‐378a (miR‐378a) content. Twenty‐five‐week‐old miR‐378a knockout Db/Db mice display preserved mt‐ATP6 and ATP synthase protein content, ATP synthase activity, and preserved cardiac function, supporting the hypothesis that miR‐378a inhibition may be a therapeutic option for maintaining ATP synthase functionality during diabetes mellitus. Evidence also suggests that long non‐coding RNAs (lncRNAs), including lncRNA potassium voltage‐gated channel subfamily Q member 1 overlapping transcript 1 (Kcnq1ot1), participate in regulatory axes with microRNAs (miRs). Prediction analyses indicate that Kcnq1ot1 has the potential to bind miR‐378a. This study aimed to ascertain whether Kcnq1ot1 interacts with miR‐378a to impact ATP synthase functionality by preserving mt‐ATP6 levels. While miR‐378a was confirmed to be significantly higher in type 2 diabetes mellitus (T2DM) patient and twenty‐five‐week‐old Db/Db mouse mitochondria (P < 0.05), mitochondrial mt‐ATP6 and Kcnq1ot1 levels were significantly reduced when compared to controls (P < 0.05). Assessments following Kcnq1ot1 overexpression in HL‐1 cardiomyocytes overexpressing miR‐378a revealed Kcnq1ot1 binding significantly reduced miR‐378a levels, as indicated by reduced luciferase activity following transfection (P < 0.05). Further, Kcnq1ot1 overexpression reduced miR‐378a levels (P < 0.05), and rescued mt‐ATP6 and ATP synthase protein content (P < 0.05). Together, these data suggest that Kcnq1ot1 and miR‐378a may act as constituents in an axis that regulates mt‐ATP6 content, and that manipulation of this axis may provide benefit to ATP synthase functionality in the heart during type 2 diabetic insult.
Machine Learning to Identify Regional and Segmental Dysfunction during Type 2 Diabetes Mellitus Progression
Authors: Rizwan Saira , Durr Andrya J. , Korol Anna , Hathaway Quincy A. , Kunovac Hathaway Amina , Taylor Andrew D. , Pinti Mark V. , Hollander John M.
Published: May 2022

Diabetes mellitus results in numerous co‐morbidities, the most serious of which is cardiovascular disease (CVD). Speckle tracking strain‐based echocardiography (STE) can be used to assess regional and segmental dysfunction prior to the onset of clinically recognizable symptoms, providing a unique opportunity to assess localized patterns of cardiac dysfunction. At present, STE has been minimally used to assess localized impacts of CVD in type 2 diabetes mellitus (T2DM). Therefore, the aim of this study was to utilize machine learning (ML) to determine the ability of STE parameters to predict T2DM and to elucidate regional and segmental impacts in a temporal fashion. Echocardiography data were collected in wild‐type and Db/Db mice at 5, 12, 20, and 25 weeks. Seventeen parameters were ­used for analyses: Complete, pulse‐wave Doppler, M‐mode, Global, Segmental, AntFree, PosteriorFree, Anterior, Posterior, Septal, Free, LatWall, PostWall, InfFreeWall, PostSeptal, and AntSeptum. Support vector machine analysis determined that STE parameters were the most accurate predictors of T2DM. Further, ReliefF feature selection was used to evaluate prevalence of each region and segment. We determined the Anterior region and AntSeptum segment to be the most prevalent (75% of the time), at weeks 5, 20, and 25 (82%, 93%, and 91% testing accuracies, respectively). The Septal region was shown to be most predictive (75% of the time), at weeks 5, 20, and 25 (84%, 93%, and 98%, respectively). Further, the InfFreeWall (76%) and LatWall segments (98%) were most predictive at 5 and 12 weeks, whereas the AntSeptum and AntFree segments (96% each) were most predictive at 20 and 25 weeks, respectively. Specifically, in some cases such as 25 weeks, the Septal region and the AntFree segment present identically, but the AntFree segment may provide a more focused target. In summary, these data demonstrate that patterns of regional and segmental dysfunction exist in the T2DM heart and can be observed in a spatiotemporal fashion using ML.
LRRK‐2 Inhibition By PF06447475 Treatment Reduces Neuronal Damage and Immune Response After Spinal Cord Trauma
Authors: Filippone Alessia , Casili Giovanna , Lanza Marika , Mannino Deborah , Basilotta Rossella , Campolo Michela , Paterniti Irene , Cuzzocrea Salvatore , Esposito Emanuela
Published: May 2022

Spinal Cord Injury (SCI) is a devastating event followed by neurodegeneration, activation of the inflammatory cascade and immune system. Dysregulated or non‐resolving inflammatory processes can affect neuronal homeostasis and drive immune cells stimulation. The leucine‐rich‐repeat kinase 2 (LRRK2) is a gene associated with the progression of Parkinson’s disease (PD), and its kinase activity was found to be upregulated after instigated inflammation of the Central Nervous System (CNS) and immune system activation. Here, we aimed to investigate the efficacy of PF‐06447475, an LRRK2 inhibitor, by counteracting pathological consequences of spinal cord trauma. The in vivo model of SCI was induced by extradural compression of the spinal cord at T6‐T8 levels, then mice were treated with PF‐06447475 (2.5‐5 and 10 mg/kg o.s) 1 and 6 hrs after SCI. We found that PF‐06447475 treatments at the higher doses (5 and 10 mg/kg) showed great abilities to reduce the degree of spinal cord tissue injury, glycogen accumulation, and demyelination of neurons associated with trauma. In addition, cytokines expression levels including interleukins (IL‐1, IL‐6, IL‐10 and12),interferong(IFN‐g)and tumor necrosis factor‐α (TNF‐α secreted and released by immune cells after trauma were decreased by LRRK‐2 inhibitor treatments. Moreover, the accumulation of CD4+and CD8+cells throughout the spinal cord lesion site of SCI mice was reduced by PF‐06447475 oral administration at the dose of 10 mg/kg. Taken together, our results suggest that exist a mutual correlation between LRRK2, neurodegeneration and immunity and that LRRK2 inhibition could have direct effects on the intervention of neuroinflammatory disorders.
Iron Deposition in the Spleen in a Murine Model of Acute Radiation Syndrome
Authors: Rittase Bradley , Slaven John E. , Muir Jeannie M. , Lee Sang‐Ho , Rusnak Milan , Brehm Grace , Symes Aviva , Day Regina
Published: May 2022

Total body radiation (TBI) can result in death associated with hematopoietic insufficiency. Although radiation causes apoptosis of white blood cells, red blood cells (RBC) undergo hemolysis due to hemoglobin denaturation. RBC lysis post‐irradiation results in the release of iron into the plasma, producing a secondary toxic event following TBI. We investigated the impact of radiation‐induced iron release on the spleen following TBI and the effects of the radiation mitigator captopril. RBC and HCT were reduced within 7 days, with the nadir at ~14 days post‐TBI. Prussian blue staining revealed ~20‐60‐fold increased Fe3+ in the spleen 7‐14 days post‐irradiation. Increased iron was associated with altered expression of iron binding and transport proteins, determined by qPCR, western blotting, and immunohistochemistry. Captopril did not prevent iron deposition in the spleen, and did not significantly modulate most iron‐binding proteins. Spleen volumes were markedly decreased 7‐14 days post‐irradiation, correlating high Fe3+ levels. At this time point, caspase‐3 was activated and we identified four additional markers of ferroptosis, an iron‐dependent programmed cell death. Interestingly, we did not observe increased expression of p21/Waf1, a marker of accelerated senescence. We investigated the effects of radiation and Fe3+ on the J774.A1 murine macrophage cell line in culture. Radiation induced p21/Waf1 and ferritin within 24 h. Radiation alone and with iron upregulated several markers of M1; radiation + iron also modulated a marker of M2 polarization. Our data indicate that following TBI, iron is deposited in the spleen where it modulates the expression of iron binding proteins and triggers ferroptosis.