bluuebunny/crossref_metadata_2025 huggingface dataset:
Non‐invasive Neuromodulation Using Ultrasound
Authors: Vasan Aditya , Friend James
Published: April 2020

The discovery of light sensitive proteins led to the development of optogenetics and allowed the precise manipulation of neuronal and non‐neuronal cells. This technique uses particular wavelengths of light to manipulate both neuronal and non‐neuronal cells
[2,
5]. It has enabled the study of information processing in the brain and recent studies are looking into clinical translational applications of the technique
[1]. Despite the advantages offered by optegenetics, it has the drawback of being invasive as light does not penetrate skin past a few millimeters. It is not possible to target deep tissue within the body using this technique and this limits the application in the context of diseases such as Parkinson’s
[3] or heart malfunction
[4].

We are developing devices that can non‐invasively manipulate cells using a technique termed “sonogenetics”. This technique uses a combination of engineering cells to be more sensitive to mechanical stimuli as well as developing ultrasound transducers capable of stimulating cells in freely moving, awake mice. We demonstrate the development of miniature transducers made from non‐hysteretic single crystal lithium niobate to be used for the development of sonogenetics. In addition, we o er mathematical insights to the mechanism of action of ultrasound on neurons. Our analysis of membrane deflection enables us to predict cellular activity based on the applied ultrasound stimulus. By extension, we use this analytical framework to inform the development of transducers and stimulation parameters for achieving non‐invasive stimulation of cells.

Support or Funding Information

WM Keck Foundation

National Institute of Health

Membrane Deflection and Capacitance Change due to Ultrasound

a–d) Membrane deflection due to an ultrasound stimulus, with parameters 7 MHz and 1 MPa pressure. The stimulus is provided for 10 ms, the resulting area change(e) and the capacitance (f) are then calculated from the input parameters. The value of capacitance generated in this model is then used in a Hodgkin‐Huxley neuron model to determine action potential generation.

Figure 1

a) LN transducer housed in a MMCX coaxial connector designed for long term behavioral studies in awake, freely‐moving mice. b) Device mounted on a mouse skull to be used for characterization using an automated hydrophone setup (c). d)Pressure profile over the transducer face with a 450 mV input results in up to 2 MPa pressures. e) Transducer attached to mouse skull for long term behavioral studies.

Figure 2
SPARC: Wireless Fecobionics Device in dogs
Authors: Wang Yanmin , Gregersen Hans , Kassab Ghassan
Published: April 2020

Objective

We developed a novel wireless device (Fecobionics) for mapping colonic and anorectal neuromuscular function. Fecobionics measures pressures, cross‐sectional areas and bending of the device and transmits data wireless to a receiver unit outside the body (figure
1). This study evaluated the safety of the device in dogs relative to the predicate THD SensyProbe device for biofeedback. Furthermore, physiological data were obtained with Fecobionics.

Methods

Twelve male Mongrel dogs with 25kg body weight were assigned into two groups: Fecobionics group and predicate control group (N=6 of each). Fecobionics and the predicate device were transanally inserted into rectum. Further, each group was divided into two sub‐groups: The acute group in which the animals were euthanized immediately after the evacuation of the device, and the chronic group in which the animals were terminated after 2 weeks. The animals were monitored daily. Tissue from different colon segments and anorectum were collected after the dogs were euthanized. HE staining and immunofluorescence staining were performed.

Results

All procedures were performed successfully without complications. All chronic animals showed normal food intake and behavior after the procedure. Wireless transmission of Fecobionics data were without significant data loss or data error. Fecobionics studies demonstrated defecation pressures in the range 60–100cmH2O. The bending of the device, representative of the anorectal angle, was less than 30 degrees. The shape of the bag was obtained from the serial cross‐sectional area measurements. The shape of Fecobionics was cylindrical in rectum and changed to hourglass shape when it passed through the anal canal. No signs of inflammation were identified in the tissue sections examined histologically or immunohistochemically (macrophages and caspase‐3) in both Fecobionics and control groups.

Conclusions

Wireless Fecobionics is as safe as the predicate device, without inducing macro‐ or microscopic changes or inflammatory status of anorectal muscle. Novel physiological data were obtained. Dogs seems not to have an anorectal angle like humans, which makes defecatory efficacy more dependent on intrarectal pressure and anal sphincter relaxation compared to humans.

Support or Funding Information

Funding

NIH SPARC OT2OD025308

The Fecobionics device (left), the wireless receiver (middle) and the data collection device (right).

Figure 1
A 3D Printable model of pulmonary arteriovenous malformations
Authors: Olson Holly , Rochon Paul
Published: April 2020

Pulmonary arteriovenous malformations (PAVMs) are vascular anomalies in which there is a direct connection between pulmonary arteries and veins, bypassing the lung capillary bed
(1). The most common cause of PAVMs are congenital, resulting from an autosomal dominant disorder, hereditary hemorrhagic telangiectasia (HHT), causing mutations in proteins important in the vascular signaling pathway
(2). Complications of untreated PAVM include stroke, hypoxemia, dyspnea, platypnea, infections, and anemia
(1,
3). Treatment of PAVMs is most commonly done so via endovascular embolization
(1). When HHT patients develop PAVMs and come to the IR clinic to obtain treatment, they are often anxious about their diagnosis and explaining the anatomy, pathology, and treatment of their disease can present challenges to the physician. The current method of patient education uses the patient’s 2D CT scans along with the physician’s narrative, which the patient may find difficult to follow because they are not trained in reading such medical datasets. We hypothesized that having a patient interact with a 3D‐printed model of PAVM would improve their understanding of the disease and anatomy involved, and decrease their anxiety regarding their diagnosis and treatment. To test this hypothesis, we first created a 3D model using the software 3D Slicer (
www.slicer.org) to segment CT scans from a single patient with multiple, untreated PAVMs. One PAVM was chosen to be segmented for its distinctive feeding artery exiting the pulmonary trunk into these fistula and distinctive vein exiting the fistula and entering the left atrium. The 3D model was then imported into Maya where it was sculpted, artistically adjusted, and enhanced to provide anatomically accurate representation of the chest anatomy applicable to this disease, to then be printed. This novel model of a disease has the potential to decrease patient anxiety prior to the treatment of their PAVM and improve their knowledge on the anatomy and pathology. The effects of HHT patient interaction with the model on the patient’s anxiety levels and understanding of the disease remains to be investigated.

CT axial section of PAVM used for segmentation

Figure 1

3D model produced in 3D Slicer

Figure 2
Radiofrequency Renal Denervation (RF‐RDN) Ameliorates the Severity of Heart Failure with Preserved Ejection Fraction (HFpEF)
Authors: Hidalgo Hunter A. , Gao Juan , LaPenna Kyle , Haydel Amelia , Kapusta Daniel , Goodchild Traci , Lefer David
Published: April 2020

Background

Heart failure with preserved ejection fraction (HFpEF) is a complex heterogeneous syndrome that results in significant morbidity, repeat hospitalizations, and excessive healthcare expenditures. At present, there are no FDA approved therapies for HFpEF patients. Subtypes of HFpEF have recently been characterized, defining these presentations using phenomapping methodologies. One subtype, termed obesity‐cardiometabolic HFpEF, demonstrates global sympathetic nervous system hyperactivity and metabolic disturbances. The role of the kidney in the pathophysiologic development of these disturbances is significant, suggesting that renal‐based therapies may prove beneficial in HFpEF. Renal denervation (RDN) has been shown to lower blood pressure in the setting of resistant hypertension.

Methods

We investigated obese ZSF‐1 rats (n = 8–10 rats/study group) that underwent either radio‐frequency renal denervation (RF‐RDN) or a sham RF‐RDN surgery. The rats were enrolled at 16 weeks of age and RF‐RDN or sham surgery was performed when the rats were 20 weeks of age followed by a 6 week observation period. We investigated a spectrum of clinically relevant parameters were assessed, including echocardiographic, radiotelemetry, exercise capacity, vascular function, cardiac fibrosis, and metabolic profiling (i.e. glucose tolerance, insulin sensitivity, serum lipids, and obesity).

Results

Rats undergoing RF‐RDN treatment exhibited significant improvements in left ventricular (LV) diastolic function according to echocardiographic determination of E/E’ ratio (Figure
). RF‐RDN treatment resulted in a significant improvement in exercise capacity (Figure
). Radiotelemetry recordings shows a significant reduction in systolic blood pressure and a halt in the progression of hypertension (Figure
). Histological evaluation of the hearts revealed attenuation of cardiac fibrosis following RF‐RDN treatment. RF‐RDN therapy failed to exert beneficial effects in terms of obesity, diabetes, or dyslipidemia in the ZSF‐1 obese rat.

Conclusion

Our findings suggest that RF‐RDN may be a viable option for the treatment of HFpEF by lowering blood pressure, improving cardiac function, and increasing exercise capacity. These findings suggest that this treatment may improve the quality of life in the setting of clinical HFpEF.

Support or Funding Information

National Institutes of General Medical Sciences (NIGMS) COBRE grant no. P30GM106392 to Dr. Kapusta

A. Diastolic functional measure of E/E’ ratio six weeks after RF‐RDN or sham RF‐RDN surgery. B. Exercise capacity duration six weeks after RF‐RDN or sham RF‐RDN surgery. C. Weekly conscious systolic blood pressure recordings. (*p < 0.05; **p < 0.01)

Figure 1
Characterizing mucus‐based biofilms in human Clostridium difficile infection
Authors: Engevik Melinda A. , Danhof Heather A. , Auchtung Jennifer , Endres Bradley T. , Bassères Eugénie T. , Garey Kevin T. , Britton Robert A. , Versalovic James
Published: April 2020

Background

Clostridium difficile infection (CDI) is a major nosocomial disease associated with antibiotic use throughout the globe. Limited therapies are available for the treatment of CDI. As a result, understanding the mechanisms of CDI and developing novel therapeutics is a high priority. C. difficile has been observed in the MUC2 mucus layer of mice and patients, indicating that C. difficile may establish a mucus‐related niche and interact with members of the mucus‐associated microbiome. We hypothesized that certain mucus‐associated microbes would promote C. difficile colonization and biofilm formation.

Methods & Results

To address this hypothesis, we utilized a human centered approach. We found that C. difficile adhered to O‐glycan structures on human MUC2 using mucin‐producing cells lines and human colonoid monolayers. To identify other microbes residing with C. difficile in the mucus layer during infection, we seeded bioreactors with healthy human stool, added antibiotics and C. diffiicle to mirror patient infection. We then inserted human MUC2‐coated coverslips and examined the community structure. 16S rRNA sequencing revealed a unique biofilm profile with substantial co‐colonization with Fusobacterium. Chemotaxis assays revealed that motile C. diffiicle was chemoattracted to Fusobacterium nucleatum subspecies nucleatum, polymorphum, and animalis. We also observed that C. difficile co‐aggregated with all F. nucleatum subspecies; an effect that was mediated by the F. nucleatum adhesin RadD. Moreover, addition of F. nucleatum enhanced C. difficile biofilm formation as denoted by ruby red biofilm staining, crystal violet and SEM. These biofilms were more resistant to standard dispersion methods and the antibiotic vancomycin. Consistent with our biofilm findings, RNAseq data revealed significant changes in C. difficile surface adhesion and antimicrobial resistance genes following exposure to F. nucleatum metabolites. Finally, publicly available datasets and patient stool samples revealed that a subset of patients with C. difficile infection exhibited high levels of Fusobacterium OTUs. C. difficile and F. nucleatum were found to co‐localize by FISH in CDI positive adult stool and pediatric IBD stool, as well as in the mucus layer of CDI patients in surgical resections.

Conclusions

Together, these data demonstrate the unique role of mucus‐associated bacteria such as F. nucleatum in enhancing C. difficile mucus‐associated biofilms.

Clostridium difficile adheres to human intestinal MUC2 (left) and aggregates with another mucus‐associated microbe Fusobacterium nucleatum in a RadD dependent manner (right).

Figure 1
Genetic Knockout of 3‐Mercaptopyruvate Sulfurtransferase Increases Acute Myocardial Ischemia/Reperfusion Injury in Aged Mice
Authors: Xia Huijing , Li Zhen , Nagahara Noriyuki , Carnal Jean , Lefer David
Published: April 2020

Background

Hydrogen Sulfide (H2S) plays important roles in cardiovascular homeostasis. 3‐Mercaptopyruvate sulfurtransferase (3‐MST), one of the endogenous H2S producing enzymes, is abundant in the heart and localized primarily in mitochondria. We hypothesized that the H2S generated by 3‐MST is critical for cardiovascular homeostasis and plays an important role in cardioprotection.

Method

Male global 3‐MST KO and wildtype (WT) mice at 12 and 23 weeks of age (n=10–15 per group) were utilized in these studies. Initial studies were performed to evaluate exercise capacity in the 3‐MST KO and WT mice. Subsequently, mice were subjected to 45 minutes of left coronary artery ligation (MI) followed by 24 hours of reperfusion (R). Myocardial infarct size was determined by Evan’s Blue + triphenyltetrazolium chloride (TTC) staining. Blood was collected at 4 hours of reperfusion for troponin‐I measurement.

Results

The 3‐MST KO mice had significantly reduced exercise capacity compared to WT at both 12 weeks of age (Duration: 47.8 ± 7.6 vs. 70.7 ± 5.9 min; Distance: 848 ± 137 vs. 1261 ± 107 m, p < 0.05) and 23 weeks of age (Duration: 13.1 ± 1.1 vs. 31.9 ± 5.5 min; Distance: 224 ± 21 vs. 562 ± 100 m, p < 0.05), suggesting important roles of 3‐MST in cardiac and/or skeletal muscle mitochondrial function. Interestingly, following MI/R, 3‐MST KO mice at 12 weeks of age exhibited significantly reduced myocardial infarct size as well as plasma troponin‐I levels when compared to WT (Infarct /Area at Risk: 36.9 ± 3.4 % vs. 49.3 ± 3.6%, p < 0.01; Troponin‐I 10.9 ± 1.8 vs. 15.1 ± 0.9 ng/ml, p = 0.05). However, at 23 weeks of age, both myocardial infarct size (64.7 ± 3.2% vs. 57.6 ± 1.5%, p = 0.05) and plasma troponin‐I level (34.8 ± 2.1 vs. 28.7 ± 1.3 ng/ml, p < 0.05) were higher in 3‐MST KO mice compared to WT.

Conclusion

Our data demonstrate that 3‐MST plays a critical positive role in the physiological response to exercise. Furthermore, despite transient cardioprotective effects of 3‐MST deficiency at a young age, loss of 3‐MST significantly worsens MI/R injury in older animals. 3‐MST appears to play important roles in maintaining cardiac function. Further studies are underway to investigate the molecular mechanisms by which 3‐MST impacts mitochondrial function and cardiovascular homeostasis.

Support or Funding Information

NIH/NHLBI R01 HL146098‐01

Myocardial infarct size following MI/R in 3‐MST KO and WT mice.

Figure 1
New Mechanistic Insights about the Enzyme Cytochrome c nitrite reductase (ccNiR) from studies of the wild type and its variants
Authors: Shahid Shahid , Pacheco A. Andrew
Published: April 2020

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic, decaheme homodimeric enzyme that catalyzes the six‐electron reduction of nitrite to ammonia. Under standard assay conditions catalysis proceeds without detected intermediates, and it has been assumed that this is also true in vivo. However, in vitro we have found it possible to trap putative intermediates, or to release partially reduced nitrogen species such as nitric oxide, by controlling the electrochemical potential at which reduction takes place. This poster will present UV‐Vis spectropotentiometric titrations of the active site variants R103Q and H257Q, and steady‐state and stopped‐flow kinetic studies of ccNiR‐catalyzed reduction of nitrite to nitric oxide by the weak reductant N,N,N′,N′‐tetramethyl‐p‐phenylenediamine (TMPD). Wild type nitrite‐loaded ccNiR is reduced chemically or electrochemically at very high potentials (midpoint potential of 246 mV vs SHE) in a concerted 2‐electron process that forms an {FeNO}7 moiety at the active site. By contrast, the H257Q variant’s active site midpoint potential is downshifted to 125 mV vs SHE and is now a 1‐electron reduction to yield an {FeNO}6 moiety. The wild type catalyzes the reduction of nitrite to NO• by TMPD. H257Q does not appear to catalyze this reaction, which may be due to its lower active site midpoint potential or to the much slower turnover of the mutant; studies with more powerful reductants are now under way.

Figure 1
High protein diet increases kidney macrophages and accelerates polycystic kidney disease
Authors: Huang Jifeng , Hsu Jung-Shan , Saigusa Takamitsu
Published: April 2020

Background

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic kidney disease that leads to end stage renal disease. Interestingly, the disease severity is highly variable even among families with the same gene mutation, suggesting factors other than genetics may affect cystogenesis. One factor that accelerates cystogenesis in PKD rodent is a high protein diet that results in renal hypertrophy. However, the mechanism of how a high protein diet promotes cystogenesis is unknown. We have shown that renal hypertrophy triggered by unilateral nephrectomy increases kidney macrophages and accelerates cystogenesis in ADPKD mouse. Therefore, we hypothesize that a high protein diet fed in ADPKD mice leads to increased kidney macrophages and accelerates cyst growth similar to a unilateral nephrectomy model.

Methods

We used adult tamoxifen inducible Pkd1flox/flox mice with or without Cagg cre‐ER. These adult conditional Pkd1 knockout mice typically requires months to develop major kidney cysts. Three weeks after cre induction, mice were fed with either a high protein (HP: 60%), a normal protein (NP: 18%) or a low protein (LP: 6%) diet for a total of 1 and 6 weeks. Some mice were given a HP diet and treated either with liposomal clodronate (to deplete macrophage) or with PBS (IP twice a week) for a total of 6 weeks. All mice were sacrificed at the end of the experiment and kidneys were harvested for histology, cytokines and flow cytometry for kidney immune cell sorting.

Results

Pkd1 mice fed a HP diet for 6 weeks resulted in increased number of kidney resident macrophage (CD11blo, F4/80 hi) and infiltrating macrophages (CD11bhi, F4/80lo) compared to kidneys from a NP or LP diet fed mice. A HP diet resulted in increased kidney pro‐inflammatory cytokines, chemokine and developed severe kidney cysts compared to a NP or LP diet group. Pkd1 mice treated with liposomal clodronate had fewer resident and infiltrating macrophages and smaller kidneys compared to control PBS treated group. Interestingly, mice fed a HP diet for 1 week already had larger kidneys compared to those fed a LP diet, but there were no differences in kidney chemokines or tubular injury marker.

Conclusions

Renal hypertrophy triggered by a HP diet increases kidney macrophages, inflammatory cytokine production and promotes cyst growth in adult Pkd1 mice. The mechanism of how dietary protein initiates and accelerates cyst growth remains to be determined.

Support or Funding Information

NIH/NIDDK K08 DK106465, R03 DK119717 PKD Foundation 232G19a
The Role of FAT10 in Alcoholic Hepatitis Pathogenesis
Authors: Jia Yue , French Sameul W.
Published: April 2020

Background

Morphologic features of Alcoholic Hepatitis (AH) balloon cells forming Mallory‐Denk bodies and bile duct metaplasia are two morphologic features consistently being found in AH. Increased FAT10 expression may induce increasing mitotic non‐disjunction and chromosome instability leading to tumorigenesis. FAT10 expression is highly up‐regulated by pro‐inflammatory cytokines IFNγ and TNFα in all cell types and tissues.

Objective

To clarify the role of FAT10 in alcoholic hepatitis pathogenesis, we studied FAT10 expression in liver biopsy samples from patients with AH.

Methods

We use gene and protein array to detect the potential changes during alcoholic hepatitis pathogenesis. The potential target, FAT10, were tested at protein expression level by using western blot, immunofluorescent (IF) staining and quantitation. And FAT10 was also studied by using knockout animal models.

Results

The results show that FAT10 is essential to maintain the function of liver cell protein quality control and Mallory‐Denk body (MDB) formation. This is consistent with the finding with fat10−/− mice which fail to form MDBs. FAT10 also signals a switch in the 26s proteasome’s proteases to cause the proteins accumulation, overexpression, and then forming MDBs. FAT10 overexpression in AH causes an extensive change in protein content in the liver leading to balloon degeneration and MDB aggregation formation, all of which is prevented in fat10−/− mice. The pathway that increases FAT10 expression includes TNFα, IFNγ, followed by NFκB and STAT3, which were all up‐regulated in AH. Using proteins expression quantitation by measuring the intensity of immunofluorescent (IF) staining, it showed the FAT10 protein expression was 4.5 fold increased in the AH livers but not in the NASH or control livers.

Discussion

Milk thistle derivatives were used to treat FAT10 overexpression experimentally. Flavanone derivatives of milk thistle inhibit IFNγ/TNFα, NFκB and STAT3 then inhibits the expression of FAT10. NFκB is the key nodal hub of the IFNα/TNFα‐response genes. Silibinin derived from milk thistle is reported to be a powerful antioxidant and has anti‐carcinoma effects including against TNFα‐induced tumor growth. Studies on Silibinin or other milk thistle derivatives to treat AH confirms that overexpressed FAT10 with NFκB and TNFα are the major key nodal molecules in these networks.

Conclusion

Overexpressed FAT10 with over‐activated NFκB and TNFα are the major key nodal molecules in alcoholic hepatitis pathogenesis. These molecules might be potential therapeutic targets.
Phloretin Overcomes Sorafenib Resistance in Hepatocellular Carcinoma Through Targeting SHP‐1‐and STAT3 Pathways
Authors: Korashy Hesham M. , Saraswati Sarita , Abdelgadir Abdelgalil M. , Tanwer Pooja
Published: April 2020

Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer death worldwide. Sorafenib (Sor), a tyrosine kinase inhibitor, is first targeted drug approved for HCC. Although, Sor prolongs survival time with limited side effects, many HCC patients become resistant to Sor. Modulation of the Signal Transducer and Activator of Transcription 3 (STAT3) and Src homology region 2 domain‐containing phosphatase‐1 (SHP‐1) pathways are believed to mediate Sor resistance. Thus, identifying effective molecules that could revert the Sor resistance in HCC is a need. Among these promising molecules, Phloretin (PH), a dihydrochalcone flavonoid found in fruit, leaves, and roots of apple tree, possesses anticancer, antitumor, and hepatoprotective effects. However, the effects and potential mechanisms of PH remain elusive.

To test our objectives, several HCC cells were tested in vitro for sensitivity to PH, Sor or both and the apoptosis, signal transduction and phosphatase activity were analyzed. The direct role of SHP‐1 was determined using chemical inhibitor and genetic SHP‐1 siRNA knock down. In vivo studies were conducted using Sor‐sensitive and ‐resistant HepG2 and Huh7 xenografts. The animal studies were conducted in conformance with the FASEB Statement of Principles for the use of animals in research and education.

The present results showed that PH inhibited cell growth and induced apoptosis in HCC cells by upregulating SHP‐1 expression and downregulating STAT3 activity. PH activated SHP‐1 by disruption of auto‐inhibition of SHP‐1, leading to reduced p‐STAT3Tyr705 level. Inhibiting SHP‐1 expression and activity using specific inhibitor, PTP inhibitor III, or siRNA upregulates p‐STAT3 and counteracted the anti‐proliferative activity of PH. Furthermore, PH induced apoptosis in two Sor‐resistant HCC cell lines and attenuated STAT3‐dependent Sor resistance in HCCs. PH markedly inhibited tumor growth in both Sor‐sensitive and Sor‐resistant xenografts in vivo by impairing angiogenesis, cell proliferation and inducing apoptosis.

In conclusion, the current data demonstrated that PH inhibits STAT3 activity in Sor‐sensitive and ‐resistant HCCs via SHP‐1–dependent mechanism (Fig.
1). Our results clearly indicate that PH may be a potent reagent for hepatocellular carcinoma and a novel targeted therapy for further clinical investigations.

Support or Funding Information

Qatar University, Qatar National Library, and King Saud University

Graphical abstract

Figure 1
PGC1α alleviates mitochondrial dysfunction via TFEB‐mediated autophagy in acute kidney injury mice
Authors: Yuan Longhui , Yuan Yujia , Zhu Lingling , Chen Younan , Cheng Jinqiu , Lu Yanrong
Published: April 2020

Objective

Cisplatin is a widely used chemotherapy drug, and always led to nephrotoxicity via causing mitochondrial dysfunction. PGC‐1α, a regulator of mitochondrial biogenesis, has been recently reported to activate TFEB‐mediated autophagy, which could remove damaged mitochondria. However, The role of PGC1α/TFEB pathway remains unknown in AKI. Thus, we aim to investigate whether the activator of PGC‐1α (ZLN005) could alleviate mitochondria dysfunction though TFEB‐mediated autophagy in AKI mice.

Methods

Mice were administered cisplatin (16mg/kg, a single i.p. injection) to induce AKI, then treated with PBS or ZLN005 for 4 days. In vitro, HK2 cells were treated with cisplatin 5mM in the presence or absence of ZLN005.

Results

In vivo, comparing with the normal mice, BUN and Crea were significantly increased in AKI mice, while decreased after ZLN005 treatment (Fig.
1A). Additionally, the severity of tubular injury as determined by histologic examination was markedly ameliorated in AKI+ZLN005 group (Fig.
1B), indicating that the activation of PGC‐1α could alleviate kidney injury in AKI mice. Moreover, comparing with the AKI mice, mitochondrial fragmentation (many swollen, small and round mitochondria) induced by cisplatin was decreased after ZLN005 treatment (Fig.
1C). Consistent with the observation in vivo, along with the improvement of mitochondrial function (Fig.
2A–C), ZLN005 activated TFEB‐autophagy in cisplatin‐induced HK2 cells (Fig.
2D). Whereas, the protective effect of ZLN005 was largely abolished when the expression of TFEB was inhibited by RNAi of TFEB(Fig.
2E).

Conclusion

These results indicated that PGC‐1α could alleviate mitochondrial function and cisplatin‐induced kidney injury via TFEB‐autophagy pathway.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China (No. 81900666)

Figure 1Figure 2
Effects of Acute SGLT2 Blockade and Dietary NaCl on Glomerular Hemodynamics in Diabetic Rats
Authors: Thomson Scott Culver , Vallon Volker
Published: April 2020

Inhibitors of the main proximal tubular Na‐glucose cotransporter (SGLT2) mitigate diabetic glomerular hyperfiltration and are approved by FDA for slowing the progression of diabetic kidney disease. It has been proposed that SGLT2 inhibitors improve hard renal outcomes by reducing the glomerular capillary pressure gradient (ΔP) through TGF signaling, which begins with a decrease in fluid reabsorption by the proximal tubule (Jprox). However, the effect of SGLT2 inhibition on ΔP has not been measured up to now.

We studied effects of acute SGLT2 blockade (ertugliflozin) on Jprox and glomerular hemodynamics by 2‐period micropuncture in STZ‐diabetic rats fed high or low NaCl diets (HS/LS). ΔP was measured by direct capillary puncture in Wistar Froemter rats (MWF), which have surface glomeruli suitable for this purpose. ΔP was computed from tubular stop‐flow pressure in Wistar rats (Wis), which lack surface glomeruli. Hence, TGF was allowed to operate normally while measuring ΔP in MWF but rendered inoperative while measuring ΔP in Wis. SGLT2 blockade was confirmed to reduce Jprox by collecting fluid from late proximal tubules for volume and inulin clearance.

Summary and Conclusions about the diabetic kidney under conditions of moderate hyperglycemia: SGLT2 blockade acutely reduces Jprox, which leads to a decrease in glomerular capillary pressure, but only when TGF is permitted to operate normally. Baseline glomerular capillary pressure varies inversely with dietary NaCl and the impact of SGLT2 blockade applies across a wide range of dietary NaCl.

Support or Funding Information

NIDDK RO1 DK112042 Merck

Group (n)

Glucose (mg/dl)

GFR(ml/min)

ΔP (mmHg)

Jprox' (nl/min)

control

SGLT2 blocked

control

SGLT2 blocked

control

SGLT2 blocked

MWF LS (6)

344±41

4.2±0.5

3.5±0.5

41.2±1.5

33.0±1.9

MWF HS (8)

408±35

4.1±0.3

3.3±0.4

36.6±1.3

32.4±1.6

Wis LS (6)

355±41

4.8±0.4

3.8±0.5

43.6±1.9

43.7±2.4

18.9±1.3

10.2±1.1

Wis HS (9)

400±52

4.5±0.5

3.8±0.6

32.6±2.3

35.7±3.0

18.1±1.3

12.6±1.1

*P<0.01 vs control period.

**P<0.01 vs LS. Jprox’ is Jprox adjusted for single nephron GFR by ANCOVA to obtain the "primary" effect of SGLT2 blockade on the tubule.
The Molecular Mechanism of Cellular Attachment for an Archaeal Virus
Authors: Lawrence C. Martin , Hartman Ross , Eilers Brian J. , Young Mark J.
Published: April 2020

Sulfolobus turreted icosahedral virus (STIV) is a model archaeal virus with a pseudo‐T=31 icosahedral virion with a mass of ~64 mega‐Daltons. Although STIV employs pyramidal lysis structures to exit the host, knowledge of the viral entry process is lacking. We therefore initiated crystallographic and cryo‐electron tomographic (CET) studies on STIV attachment and entry. Cryoelectron micrographs showed virion attachment to pili‐like structures emanating from the Sulfolobus host. Tomographic reconstruction and sub‐tomogram averaging revealed pili recognition by the STIV C381 turret protein. Specifically, the triple jelly roll structure of C381 determined by X‐ray crystallography shows that pilus recognition is mediated by conserved surface residues in the second and third domains. In addition, the STIV petal protein (C557), when present, occludes the pili binding site, suggesting that it functions as a maturation protein. Combined, these results demonstrate a role for the namesake STIV turrets in initial cellular attachment and provide the first molecular model for viral attachment in the archaeal domain of life. The work also nicely illustrates the synergistic power of hybrid structural studies utilizing cryo‐electron tomography, single particle analysis and crystallography to model cellular structures at the molecular level.

Support or Funding Information

This work was supported by the National Science Foundation, grants DEB‐1342876 to M.J.Y. and MCB‐1413534 to C.M.L.

Tomographic structure of the STIV pilus interaction. (A–J)2.25 nm thick sections at 10 nm spacings through the tomogram. (K) Subvolume averages are shown. The virus/pilus interaction is at the viral turret.(L) The turret highlighted in K rotated 90°, looking into a pilus cross section. C381 is light, medium and dark purple showing the 1st, 2nd and 3rd domains. M)Pilus recognition is mediated by the 2nd and 3rd domains. N)Surface exposed, strictly conserved residues mediate C381/pili recognition.

Figure 1
An interbacterial toxin inhibits target cell growth by synthesizing (p)ppApp
Authors: Whitney John , Ahmad Shehryar , Wang Boyuan , Laub Michael
Published: April 2020

Bacteria have evolved sophisticated mechanisms to inhibit the growth of competitors. One such mechanism involves type VI secretion systems, which can be used by bacteria to directly inject antibacterial toxins into neighbouring cells. Many of these toxins target cell envelope integrity, but the full range of growth inhibitory mechanisms remains to be determined. Here, we discover a novel type VI secretion effector, Tas1, in the opportunistic pathogen Pseudomonas aeruginosa. A crystal structure of Tas1 reveals similarity to enzymes that synthesize (p)ppGpp, a broadly conserved signaling molecule in bacteria that modulates cell growth rate, particularly in response to nutritional stress. Strikingly, however, we find that Tas1 does not synthesize (p)ppGpp, and instead pyrophosphorylates adenosine nucleotides to produce (p)ppApp at rates of nearly 180,000 per minute. Consequently, delivery of Tas1 into competitor cells drives the rapid accumulation of (p)ppApp, depletion of ATP, and widespread dysregulation of essential metabolic pathways, resulting in target cell death. Collectively, our findings reveal a new mechanism for interbacterial antagonism and demonstrate, for the first time, a physiological role for the metabolite (p)ppApp in bacteria.

Support or Funding Information

Canadian Institutes of Health Research, David Braley Centre for Antibiotic Discovery, National Institutes of Health, Howard Hughes Medical Institute

Structure of the (p)ppApp synthetase toxin Tas1 in complex with its cognate immunity protein, Tis1.

Figure 1

Tas1 is a pyrophosphokinase that is specific for canonical 5′‐adenosine nucleotides.

Figure 2
Three Self‐resistance Modifications Discovered in Nucleoside Antibiotic Muraymycin
Authors: Cui Zheng , Van Lanen Steven G.
Published: April 2020

Background

Muraymycins, discovered by Wyeth Research in 2002, are lipopeptidyl nucleoside antibiotics that inhibit the enzyme bacterial translocase I involved in peptidoglycan cell wall. Muraymycin exhibits good activities against gram‐positive bacteria and excellent activity against a permeabilized E. coli. However, the self‐resistance mechanism during muraymycin biosynthesis has not been elucidated.

Objectives

Our long‐term research goal is to delineate the biosynthetic pathway of muraymycin in order to develop more active analogues through chemo‐enzymatic synthesis and mutasynthesis. Our current goal is to study the self‐resistance mechanism during muraymycin biosynthesis to assist structure–activity relationship (SAR) study of muraymycins.

Methods

To interrogate the self‐resistance mechanism during muraymycin biosynthesis, we analyzed its biosynthetic gene cluster. Two genes, mur28 and mur29 that potentially catalyzed the resistant modifications were cloned and expressed. We also cultured muraymycin‐producing strain to isolate resistant modified muraymycins. LC‐MS and NMR analysis was utilized to determine the resistant modified products.

Results

Mur28 and Mur29 were functionally assigned in vitro as an ATP‐dependent phosphotransferase and an ATP‐dependent nucleotidyltransferase, both modifying the 3″‐OH of muraymycins. Interestingly, from single‐substrate kinetic analyses, we discovered that Mur28 preferentially phosphorylates a hypothetical biosynthetic precursor of muraymycin: a GlyU‐ADR disaccharide, while Mur29 preferentially adenylates muraymycin. Besides the two resistant modification products, a new muraymycin acetylaed at the 5″‐amine of the ADR moiety was isolated from muraymycin‐producing strain. The adenylated, phosphorylated and acetylated products have significantly reduced MraY inhibitory activities and reduced antibacterial activities, compared with the respective unmodified muraymycins.

Conclusion

Phosphorylation, nucleotidylation and acetylation, are three types of resistant modifications that are often found as mechanisms of resistance to antibiotics, most notably aminoglycoside antibiotics. Here, we first discovered all these three types of resistant modifications in the nucleoside antibiotic muraymycin. Among these, Mur28 serves as complementary self‐resistance mechanism, with a distinct temporal order during muraymycin biosynthesis.

Support or Funding Information

This work was supported by the National Institutes of Health (grant AI087849).

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Figure 1
Oxidative Stress Induced by Loss of Superoxide Dismutases Leads to Activation of No‐Go Decay
Authors: Cianflone Emily , Champine Madison , Lobb Amber , Segal Scott P.
Published: April 2020

Reactive oxygen species (ROS) are known to cause oxidative damage in the cell and have been connected to many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and cancer. Superoxide dismutases (SODs) are a group of enzymes that act to prevent oxidative damage from ROS by converting O2− to H2O2. In Saccharomyces cerevisiae, there are two SOD proteins that are expressed, each of which is conserved in humans. Sod1p is a cytoplasmic Cu/Zn coordinated enzyme, whereas Sod2p coordinates Mn2+ and localizes to mitochondria. Although there is significant data showing that loss of SOD proteins allows for high levels of intracellular oxidation, it is unknown what effects that would have on the translating pool of mRNA. Current work has revealed that strains lacking Sod1p have a moderate decrease in general translation and an increase in P‐body assembly. Furthermore, this reduction in translation is likely due to the formation of 8‐oxo(G) bases in mRNA, which can cause ribosome stalls leading to the subsequent activation of the No‐Go quality control pathway. No‐Go decay is mediated by the proteins Dom34p and Hbs1p, which act together to remove stalled ribosomes and promote endonucleolytic cleavage of the mRNA at the stall site. The 3′ fragment of mRNA is then localized to P‐bodies to be degraded 5′ to 3′ by Xrn1p, whereas the 5′ fragment is degraded by the cytoplasmic exosome.

Sod2p performs a similar role to Sod1p, as it also converts O2− to H2O2, and plays an important role in preventing the formation of strong oxidant peroxynitrate (ONOO−). Given that it localizes to mitochondria, a loss of Sod2p would result in higher levels of mitochondrially derived ROS. To determine the effect of mitochondrially derived ROS on the translating pool of mRNA, both general translation and P‐body assembly will be analyzed in strains lacking Sod2p. If general translation is reduced and P‐body assembly is increased, then it will be ascertained whether No‐Go Decay is being upregulated in the absence of Sod2p. To perform this analysis, double mutant strains lack both Sod2p and No‐Go Decay will be created and further analyzed for general translational control and P‐body assembly.
From mathematical model towards educational simulation game of hemodynamics of cardiovascular system
Authors: Kulhanek Tomas , Kofránek Jiøí , Mateják Marek , Ježek Filip , Šilar Jan , Mládek Arnošt , Feberová Jitka , Feber Martin , Zazay Awista
Published: April 2020

Recently we have created a new open‐source technology called BodyLight.js which combines the current standard for web‐based applications including Web‐Assembly for fast computation of mathematical models, web components for building independent transferable objects, and WebGL for involving 3D graphic
[1,
2].

We have published methodology and implementation of several models of the cardiovascular system
[3].

We have been creating graphical components in 2D and recently in 3D which can be used as animations in simulation application.

There are numerous steps involved when going from an idea to a finished project. In this contribution we describe the workflow going from concept to mathematical model and computer graphics to an educational simulation application. This workflow is presented by building a simulation game of hemodynamics of the cardiovascular system.

Each step of the workflow can be executed by different domain expert. Physiologist designs concept, mathematician writes and implements mathematical models and executable model simulation programs, graphical designer produces graphical objects based on design concepts which can be animated in simulation programs, and software engineer maintains the system. However, all these professionals need to be able to understand each other and work together.

Physiologists must be able to grasp the physiological significance and to tell mathematician how to formalize the problem. Mathematicians and software engineers need to understand physiology in order to produce plausible and verifiable model simulation application. Computer graphics designers must be skilled in programming as they need to produce animated objects that can be used in simulation applications. This connection of expertise is crucial, and was already proposed e.g. by N. Wiener in his works about Cybernetics
[4].

We demonstrate that such creative connection of individuals with combined and shared expertise can produce high quality educational materials.

Support or Funding Information

MPO TRIO FV20628, MPO TRIO FV30195

Collaborative workflow designing simulator of pressure‐volume loop cycle of hemodynamics model using Bodylight.js software tools.

Figure 1
SPARC_A DNA‐based optical nanosensor for in vivo imaging of acetylcholine in the peripheral nervous system
Authors: Clark Heather , Monaghan James , Yang Hongrong , Xia Junfei , Mu Michelle , Lovely Alexander , Micovic Nicholas
Published: April 2020

The study of neurotransmitter signaling helps to understand fundamental neuronal circuits and develop strategies against neurodegenerative diseases. Acetylcholine (ACh) plays a pivotal role in modulating neuron functions. Monitoring its physiological level in real‐time and probing its biological distribution is of crucial importance. However, the current techniques are subject to temporal and spatial limitations which impede their further in vivo applications. Herein we report the development of a ACh nanosensor by using DNA as a scaffold, acetylcholinesterase as a recognition component, pH‐sensitive fluorophores as signal generator, and α‐bungarotoxin as a targeting ligand. The nanosensor was delivered to the submandibular ganglion in living mice by microinjection for in vivo imaging of endogenous ACh release. The ACh nanosensor selectively binds to acetylcholine receptors on the post‐synaptic neurons, and display a sensitive response to physiological ACh range from 0.228 μM to 358 μM in ex vivo calibration. The ACh nanosensors also respond to endogenous ACh release triggered by electrical stimulation in a reversible and dose‐dependent manner. We also found that the treatment of cholinergic inhibitor, Vesamicol, significant suppresses the response of the ACh nanosensor. This work provides a tool for in vivo imaging of ACh dynamics with unprecedented capabilities to enhance the spatial and temporal resolution of detection. We envision such a sensor platform could be applied to imaging other neurotransmitters in mammalian system as well as extended to other organ systems in the peripheral nervous system

Support or Funding Information

NIH common fund_SPARC_Project number: 1OT2OD024909‐01

DNA‐based acetylcholine (ACh) nanosensors detect the ACh in the submandibular ganglion (SMG) of living mice. (A) ACh sensors were microinjected into the SMG of a living mouse. With the targeting ability of bungarotoxin, the ACh nanosensor can immobilize in the synaptic cleft of the mouse. (B) The mechanism of ACh nanosensors. (D) ΔF/F from pHAb channel in response to electrical stimulation (indicated by red arrow).

Figure 1
Exercise‐induced Increase in Hexokinase II‐mitochondria Association Reduces Cardiac Ischemia‐Reperfusion Injury in Rats
Authors: Mishra Jyotsna , Kwok Wai-Meng , Fitts Robert H. , Camara Amadou K.S.
Published: April 2020

Introduction

Exercise‐training has been shown to modulate signaling pathways that contribute to improved mitochondrial function in the heart. AKT, a serine/threonine protein kinase pathway is a critical regulator of a variety of cellular functions and its activation is implicated in cardioprotection from ischemia‐reperfusion (I/R) injury. Importantly, the AKT pathway has been shown to be upregulated in exercise. However, the interplay between AKT signaling, mitochondrial function and cardio‐protection in exercise remains to be well elucidated.

AIM

Herein, we investigated the role of exercise‐induced AKT/Hexokinase II (HKII)‐signaling as a potential mechanism in preserving mitochondrial function and thereby, contribute to cardioprotection against I/R injury.

Methods

Isolated hearts from rats, randomly assigned to either sedentary (SED) group or 6–8 weeks treadmill trained (TRN) group, were subjected to 30 min global ischemia and 5 min reperfusion. Mitochondria were isolated immediately after reperfusion and the effects of exercise on cardiac mitochondrial bioenergetics, including changes in O2 consumption rate, mitochondrial membrane potential (ΔΨm), and mitochondrial calcium retention capacity (CRC) was examined. Western blot was performed to quantify the total AKT, p‐AKT and p‐HKII. In addition, we checked the subcellular distributions of HKII and examined the regulation of apoptotic markers at the mitochondrial level. In some hearts, cardiac functional variables and infarct size were also assessed after 120 min reperfusion in SED and TRN rats.

Results

We found that exercise increased the phosphorylation of AKT in TRN rat hearts, which was associated, concomitantly, with increased phosphorylation of HKII and increased translocation to mitochondria following IR. TRN rats also showed a decreased expression of Bax and DRP1 in mitochondrial fraction and inhibition of caspase‐3 activation. Furthermore, exercise preserved the mitochondrial bioenergetics with significantly higher respiratory control index and more normalized ΔΨm compared to SED rats (see schematic). CRC was greater in TRN rats compared to SED rats (226.6 ± 13.3 nmol /mg protein vs 173.3 ± 13.3 nmol /mg protein). These improvements in mitochondrial function in TRN rats correlated with improved cardiac function during reperfusion.

Conclusion

The results indicate that AKT‐mediated HKII‐mitochondrial interaction due to exercise protects mitochondria by inhibiting mitochondrial apoptotic cascades and likely contributes to the increased cardioprotection against IR injury.

Support or Funding Information

This work was supported by NIH grant R01 HL131673‐01A1 to AKSC and WMK

Schematic diagram for exercise‐induced activation of AKT‐HKII signaling pathway during IR injury (A). Time course of the mitochondrial O2 consumption, membrane potential (B) and calcium retention capacity (C) in SED and TRN rats after IR stress.

Figure 1