bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel CRISPR/Cas9 Knock‐in Rat Model Defines A Polymorphic Variant of Secreted Phosphoprotein 2 as A Quantitative Trait Nucleotide Linked to The Heritability of Blood Pressure
Authors: Chakraborty Saroj , Nie Ying , Cheng Xi , Mell Blair , Galla Sarah , Joe Bina 
Published: November 2023

Hypertension is a complex polygenic disease caused by a combination of inherited (genetic) and environmental factors. Rat models of inherited hypertension serve as tools to dissect and prioritize genetic factors as candidate genes causing hypertension. One such candidate gene prioritized through systematic linkage and substitution mapping is Spp2 or Secreted Phosphoprotein 2. A single non‐synonymous G/T polymorphism between the Dahl Salt‐ Sensitive (S) rats and Spontaneously Hypertensive Rats (SHR) at the Spp2 locus was hypothesized to cause a reduction in blood pressure (BP) observed in the S.SHR congenic strain spanning the Spp2 locus. To test this hypothesis, a novel rat model was generated using the CRISPR/Cas9 precision‐engineering technology, whereby the ‘G’ allele at the Spp2 locus of the S rat was replaced by the ‘T’ allele of the SHR rat. Protein modeling prediction by SWISSPROT indicated a significantly altered protein structure of the Spp2 protein in the resultant Spp2 knock‐ in rescue model. Following transgenesis, pups born were genotyped and grouped into founders and non‐founders. The founder and non‐founder rats were isogenic except for the Spp2 locus, wherein the founder S rats contained the ‘G’ allele and the non‐founder S rats contained the ‘T’ allele. Both founder and non‐founder rats (n=12 male rats/group) were fed a high salt (2% NaCl) containing diet and their BP was monitored by radiotelemetry. Both systolic and diastolic BP of the Spp2 knock‐in rescue model were significantly lower compared to that of the non‐founder S rats. These data provide conclusive evidence for a single nucleotide polymorphism within the Spp2 gene as a quantitative trait nucleotide (QTN) responsible for the inheritance of blood pressureSupport or Funding InformationFunding for this work to BJ from the NHLBI/NIH (HL020176) is gratefully acknowledged
Activation of collecting duct NOS1β during high salt feeding is critical for down‐regulation of Na+ and K+ transporters, in part, via down‐regulation of aldosterone
Authors: Hyndman Kelly A , Collins Jessika D , Pollock Jennifer S 
Published: November 2023

We determined that deletion of nitric oxide synthase‐1β (NOS1β) from the collecting duct (CD) results in inappropriate ENaC activation during high salt diet. Given that sodium influx via ENaC is a driving force for potassium (K) secretion, we hypothesized that CD NOS1β regulates potassium handling. Male control and CDNOS1KO mice were placed on either 1) 0.4% NaCl + 1% K diet, NSNK, 2) 4% NaCl + 1% K diet, HSNK, 3) 0.4% NaCl + 0% K diet, NSLK, or 4) 4% NaCl + 0% K diet, HSLK, for 3 – 4 days. Food and water intake was similar between genotypes. As expected, plasma potassium was similar between NSNK (control 4.8 ± 0.3; KO 4.6 ± 0.2 mM) and HSNK (control 4.6 ± 0.4; KO 4.6 ± 0.1 mM), regardless of the genotype; however it was significantly reduced in the NSLK (control 3.9 ± 0.2; KO 3.5 ± 0.1 mM) and HSLK treatments (control 3.5 ± 0.1; KO 4.1 ± 0.2 mM). Plasma aldosterone failed to be suppressed in the CDNOS1KO mice on HSNK diet (control 74 ± 5; KO 422 ± 64 pg/ml, P < 0.05). Interestingly, plasma renin concentration was appropriately suppressed in both genotypes on HSNK diet. However, plasma aldosterone was appropriately suppressed in both genotypes on the potassium deficient diets (NSLK: control 81 ± 41, KO 177 ± 48 pg/ml; HSLK: control 95 ± 25, KO 100 ± 32 pg/ml). CDNOS1KO mice given spironolactone pellets (50 mg/kg b.w.) for 7 days while on a HSNK diet presented with significantly higher plasma potassium compared to controls with similar treatment (4.2 ± 0.07 mM vs. 3.9 ± 0.1 mM, P = 0.04). Western blot analysis of renal cortical lysates determined that during HSNK feeding CDNOS1KO mice display increased Kir4.1 potassium channel expression, increased NCC activation, increased NKCC2 expression, and ENaC activation compared to control cortical lysates. These data suggest that activation of NOS1β in the CD during high salt feeding modulates not only sodium handling but also potassium handling, in part, via down‐regulation of aldosterone.Support or Funding InformationResearch reported in this publication was supported by NIH NIDDK under award number K01DK105038 to K.A.H. and NIH NHLBI under award number P01HL95499 to J.S.P.
Cyclophosphamide Treatment Attenuates Hypertension in an Experimental Model of Systemic Lupus Erythematosus
Authors: Wolf Victoria L. , Ryan Michael J. 
Published: November 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease that predominantly affects women during their reproductive years, leading to speculation that estrogens factor in the pathogenesis of the disease. However, published data from our laboratory show a complex temporal relationship with estrogens likely serving a permissive role for SLE disease early in life, but acting to protect against cardiovascular risk factors like hypertension during adulthood. Hypertension, a major cardiovascular risk factor and independent predictor of mortality in women with SLE, is prevalent in these patients for reasons that remain unclear. Our laboratory showed that autoimmunity is an important factor driving the pathogenesis of hypertension and that immunosuppressive therapies can attenuate the development of hypertension in an experimental mouse model of SLE. Cyclophosphamide is a common immunosuppressive treatment for patients with SLE that works by inhibiting DNA synthesis via the alkylation of nucleic acids in rapidly dividing cells. While CYC‐based treatment regimens have significantly improved survival rates, they also contribute to an increased prevalence of premature ovarian failure among patients treated for advanced lupus nephritis that could ultimately impact estrogen status of these women. We hypothesized that the administration of CYC (25 mg/kg, once/week, IP injection) for four weeks would blunt the development of hypertension and renal disease in an established female mouse model of SLE with hypertension (adult NZBWF1 female mice at 30 weeks of age), and that the estrogen status of the mice would be impaired as a side effect of the treatment. Mean arterial pressure (MAP) was measured by indwelling carotid catheters at the conclusion of the study in conscious freely moving mice. Results suggest that MAP was lower in CYC treated SLE mice (n=4) compared to vehicle treated SLE mice (n=4) [119.7±2.0 vs 135.1±5.1 mmHg, p<0.05]. Preliminary data suggest that treatment with CYC attenuates renal injury assessed by urinary albumin excretion [0.0858±0.0657 (n=4) vs 0.4539±0.2565 (n=6) mg/day] and may have had an effect to reduce uterine weight [0.13±0.01 (n=5) vs 0.20±0.03 (n=6) % of body weight], a surrogate marker for estrogen status. CYC treatment also did not significantly affect body weight, with a decrease of 2.68±0.02% (n=5) in CYC treated SLE mice compared to a decrease of 2.88±0.01%(n=6) in vehicle treated SLE mice, suggesting that the change in MAP was not caused by the loss of body weight that sometimes accompanies cytotoxic drugs. Additional studies are required to determine the impact of CYC on hormonal status in mice with SLE and whether hormone therapy can be safely used in SLE patients using CYC that may be at risk of losing ovarian function.Support or Funding InformationVA Merit Award (BX002604‐01A2) NIH (PO1HL051971, P20GM104357, T32HL105324‐05)
Is estrogen an inhibitor of sodium appetite and an enabler of sodium skin reservoir?
Authors: Mecawi Andre Souza , Menezes Veronica Cristina Lopes , Dos‐Santos Raoni Conceição , Monteiro Livia Rocha Natalino , Paes‐Leme Bruno , Casartelli Evelton Alves , Cortes Wellington Silva , Vivas Laura , Reis Luis Carlos 
Published: November 2023

It was demonstrated that estradiol could modulate neuroendocrine and behavioral responses concerning body fluid and cardiovascular homeostasis. Not entirely consensual data support that estrogen may influence sodium appetite and food intake in cycling female and estrogen‐replaced ovariectomized (OVX) rats as well. The key question herein is whether differences in the baseline or stimulated water and sodium intake are a function of circulating estrogen in OVX rats and if there are any relationship with a conceivable skin sodium storage. Female Wistar (~230 g) rats were bilaterally ovariectomized and injected for 7 days with oil (OVX) or estradiol cypionate, 40 μg/kg (OVX+40). We evaluated spontaneous and induced water and sodium intake, plasma and urine electrolyte concentration. In addition, sodium skin level was determined in replete and sodium‐depleted OVX and OVX+40 rats. OVX rats displayed higher food intake compared to estrogen‐replaced rats. OVX+40 rats also showed a significant increase in sodium intake (7%) and normalized plasma sodium 24 h after sodium depletion, while OVX rats still maintained hyponatremia (−4%). However, OVX+40 rats expressed reduced sodium intake induced by sodium depletion compared with OVX rats. Estrogen‐replaced rats presented higher skin stored sodium load both at baseline (24%) and at 24 h after sodium depletion (52%). The osmotically active sodium load stored on the skin was higher (3.5%) in the estrogen‐replaced rats at baseline, but decreased (8%) after sodium depletion in this same group. The osmotically inactive sodium was not changed at any condition. The sodium/water ratio was significantly higher in the group of estrogen‐replaced rats after sodium depletion, compared to the OVX rats without replacement. Our data clearly show that estrogen influence behavioral ingestive response after sodium depletion in OVX rats. Regarding the higher plasma levels in our experimental paradigm, we may suggest that estrogen replacement makes them less vulnerable to volume depletion. This condition is possible based on, at least in part, on the ability of estrogen replacement to establishing higher levels of sodium stored in the skin.Support or Funding InformationCNPq, FAPERJ
Assessment of Hydration State by Combining Urine Color and Void Number
Authors: Adams J.D. , Johnson Evan C , Jansen Lisa T , Mauromoustakos Andy T , Perrier Eric , Kavouras Stavros A 
Published: November 2023

PURPOSETo evaluate the diagnostic ability of two combined practical markers for elevated urine osmolality in free‐living adults.METHODSOne hundred and one healthy adults (females N=52, 1.64±0.06 m, 72.0±16.6 kg, 26.9±6.1 kg/m2; males N=49, 1.77±0.07 m, 81.5±16.0 kg, 26.0±4.7 kg/m2) collected urine for 24 h on two occasions day 1 (D1) and day 2 (D2), separated by 1 week. Urine was analyzed for urine osmolality (UOsm), color (UC), while the number of voids (Void) was also recorded. Receiver Operating Characteristic (ROC) analysis was performed for UC, Void, and combination of UC and Void, to determine markers' diagnostic ability for detecting high urine osmolality (UOsm >500 mmol·kg−1).RESULTSFor the first and second day, UOsm was 415±216 & 457±225 mmol·kg−1, UC was 2±1 & 2±1, and Void number was 8±3 & 8±3. ROC analysis results for UC for UOsm>500 mmol·kg−1 both D1 and D2 had good overall classification ability (area under the curve, AUC 81–85%), with good sensitivity (77–81% and good specificity (76–80%). Threshold analysis revealed a UC > 3 for both D1 and D2. ROC analysis results for Void for D1 and D2 had good overall classification ability (AUC both 72%), with good sensitivity (78–80%), but moderate specificity (67–74%). Threshold analysis revealed a void number > 8 for D1 and > 7 D2. Based on the ROC analysis, combining both UC and Void number displayed good overall classification ability for D1 and D2 (AUC 84–86%) with moderate sensitivity (69–78%), and good specificity (89–90%).CONCLUSIONBoth urine color alone and the combination of urine color with void number can be used as a valid and simple diagnostic measure to detect elevated urine osmolality.Support or Funding InformationThe study was supported by a grant by Danone Research
ALDOSTERONE INFUSION INTO THE 4TH VENTRICLE PRODUCES ACTIVATION OF HSD2 NEURONS IN THE NTS AND SODIUM APPETITE
Authors: Gasparini Silvia , Melo Mariana Rosso , Andrade‐Franze Glaucia Maria Fabricio , Menani Jose Vanderlei , Colombari Eduardo 
Published: November 2023

AimAldosterone sensitive neurons characterized by the presence of the enzyme HSD2 are present in the nucleus of the solitary tract (NTS) near to the 4th V. These neurons are activated under various conditions that stimulate sodium intake, suggesting that they may have a role in sodium homeostasis. A recent study showed that chronic infusion of low dose of aldosterone into the 4th ventricle (4th V) induces sodium intake. Indeed, in the present study, we sought to investigate if the dose of aldosterone that induces sodium intake when infused into the 4th V also activates HSD2 neurons in the NTS. In addition, it was also analyzed the activation of other areas of the brain important for the control fluid‐electrolyte balance and directly or indirectly connected with the NTS, like the lateral parabrachial nucleus (LPBN), central amygdala (CeA), supraoptic nucleus (SON), paraventricular nucleus (PVN) and medial preoptic nucleus (MPO).Material and MethodsMale Holtzman rats with stainless steel cannulas implanted into the 4th V were used. Rats had free access to water and food and also to 1.8% NaCl during 2 hours/day. Water and 1.8% NaCl intake was measured in the first 5 days of infusion of vehicle or aldosterone (100 ng/μl/h) into the 4th V. On the sixth day of infusion, the brains were removed and the number of neurons expressing c‐Fos or c‐Fos and the enzyme HSD2 were quantified in different areas of the brain by immunohistochemistry.ResultsChronic infusion of aldosterone (100 ng/h) into the 4th V increased 1.8% NaCl intake (10.3 ± 3, vs. vehicle: 2.3 ± 1 ml/2 h), without changing water intake. The infusion of aldosterone into the 4th V increased c‐Fos expression in HSD2 neurons in the intermediate NTS (3 ± 1, vs. vehicle: 0 ± 0 cells/section, p < 0.05) and rostral NTS (7 ± 2 vs. vehicle: 0 ± 0 cells/section, p < 0.05). The expression of c‐Fos also increased in non HSD2 neurons in the commissural NTS (22 ± 6, vs. vehicle: 5 ± 3 cells/section, p < 0.05). Aldosterone into the 4th V also increased c‐Fos expression in the SON (21 ± 2, vs. vehicle 7 ± 2 cells/section, p < 0.05), CeA (29 ± 9, vs. vehicle: 0 ± 0 cells/section, p < 0.05), without changing c‐Fos expression in the LPBN (14 ± 4, vs. vehicle: 13 ± 5 cells/section, p > 0.05). PVN (23 ± 5, vs. vehicle: 10 ± 4 cells/section, p > 0.05) or MPO (22 ± 5, vs. vehicle 7 ± 7 cells/section, p > 0.05.ConclusionThe activation of HSD2 neurons in the NTS by aldosterone infused into the 4th V probably activates a central circuitry involved in the stimulation of sodium intake that may include one or more areas like SON and CeA.Support or Funding InformationPROPE‐UNESP; CAPES; CNPq; FAPESP
DNA origami supported precision measurements of biomolecular interactions and structure
Authors: Dietz Hendrik 
Published: November 2023

Programmable self‐assembly with DNA origami allows creating custom‐shaped nanoscale objects. Through this capacity, DNA origami enables constructing custom instruments to perform precision measurements of molecular interactions and structure, with enhanced control over positioning, orientating and manipulating the molecules under study. In my presentation I will report about a series of experiments in which we exploited this capacity to dissect the weak stacking forces between individual basepairs (1) and the forces that act between pairs of nucleosomes (2).In experiment (1), we directly measured at the single‐molecule level the forces and lifetimes of DNA base‐pair stacking interactions for all stack sequence combinations. Our experimental approach combined dual‐beam optical tweezers with DNA origami components to allow positioning of blunt‐end DNA helices so that the weak stacking force could be isolated. Base‐pair stack arrays that lacked a covalent backbone connection spontaneously dissociated at average rates ranging from 0.02 to 500 per second, depending on the sequence combination and stack array size. Forces in the range from 2 to 8 piconewtons that act along the helical direction only mildly accelerated the stochastic unstacking process. The free‐energy increments per stack that we estimate from the measured forward and backward kinetic rates ranged from −0.8 to −3.4 kilocalories per mole, depending on the sequence combination. Our data contributes to understanding the mechanics of DNA processing in biology, and it is helpful for designing the kinetics of DNA‐based nanoscale devices according to user specifications.In experiment (2), we performed a direct measurement of inter‐nucleosomal interactions by integrating two nucleosomes into a DNA origami‐based force spectrometer, which enabled sub‐nanometer resolution measurements of nucleosome‐nucleosome distance frequencies via single particle electron microscopy imaging. From the data we derived the Boltzmann‐weighted distance‐dependent energy landscape for nucleosome pair interactions. We find a shallow but long‐ranged (~6 nm) attractive nucleosome pair potential with a minimum of −1.6 kcal/mol close to direct‐contact distances. The relative nucleosome orientation had little influence, but histone H4 acetylation or removal of histone tails drastically decreased the interaction strength. Due to the weak and shallow pair potential, higher‐order nucleosome assemblies will be compliant and experience dynamic shape fluctuations in the absence of additional co‐factors. Our results contribute to a more accurate description of chromatin, while our force spectrometer provides a powerful tool for the direct and high‐resolution study of molecular interactions using imaging techniques.Support or Funding Information
Playing a tug of war with integrin and Notch receptors
Authors: Ha Taekjip 
Published: November 2023

It is now widely appreciated that cancer cells and stem cells can change their cell fate (differentiation, metastasis, etc.) depending on their mechanical environment. Mechanical sensing is likely to be initiated by individual membrane receptor proteins that are in direct contact with the mechanical environment. In order to understand how molecular level mechanical events trigger a cellular response, we need to examine the forces applied across individual cellular proteins during mechanical signaling.To define the single molecular forces required to activate signaling through a ligand‐receptor bond, we developed the tension gauge tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor‐ligand tension. Using a range of tethers with tunable tension tolerances, we showed that cells apply a peak tension of about 40 picoNewtons (pN) to single integrin‐ligand bonds during initial adhesion. This tension value was consistent across different cell types. We then presented a mixture of weak and strong TGTs (12 pN and 54 pN nominal tension tolerance) to the cells in order to better mimic native cellular environments that are likely heterogeneous. Surprisingly, just one or two strong TGTs were sufficient to allow the cells to adhere as long as there is a high density of weak tethers. Therefore, mechanical tugging through one or two integrins is enough to convince the cell that the substrate is stiff enough for adhesion. Even more surprisingly, when we presented a mixture of two different weak TGTs (12 pN and 23 pN), cells adhered to the surface despite the fact that individually neither of the two tethers could support cell adhesion. Furthermore, we found that the 23 pN tether can exert its influence even at very high dilution, down to as few as two tethers per cell. This phenomenon of ‘ultrasensitivity in differential force sensing through integrin’ can be explained by a model where simultaneous rupture of weaker bonds exerts a transient high tension across a single integrin to activate that integrin.Notch signaling, involved in development and tissue homeostasis, is activated at the cell‐cell interface through ligand‐receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. In our original TGT paper, we showed that Notch signaling is activated even for the weakest TGT (12 pN), suggesting that if Notch activation requires force, it must be less than 12 pN. Subsequently, the Blacklow lab show that the exposure of a Notch cleavage site required for its activation occurs at a tension of ~5 pN39, consistent with our estimate of a 12 pN upper limit. We recently developed LTGT (a low tension gauge tether) utilizing the low unbinding force of ~ 4 pN between single‐stranded (ss)DNA and E. coli ssDNA binding protein (SSB) and showed that Notch activation requires forces between 4 and 12 pN.
A 3D code in the human genome
Authors: Aiden Erez Lieberman 
Published: November 2023

Stretched out from end‐to‐end, the human genome – a sequence of 3 billion chemical letters inscribed in a molecule called DNA – is over 2 meters long. Famously, short stretches of DNA fold into a double helix, which wind around histone proteins to form the 10nm fiber. But what about longer pieces? Does the genome's fold influence function? How does the information contained in such an ultra‐dense packing even remain accessible?In this talk, I describe our work developing ‘Hi‐C’ (Lieberman‐Aiden et al., Science, 2009; Aiden, Science, 2011) and more recently ‘in‐situ Hi‐C’ (Rao & Huntley et al., Cell, 2014), which use proximity ligation to transform pairs of physically adjacent DNA loci into chimeric DNA sequences. Sequencing a library of such chimeras makes it possible to create genome‐wide maps of physical contacts between pairs of loci, revealing features of genome folding in 3D.Next, I will describe recent work using in situ Hi‐C to construct haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify □10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind the protein CTCF. The CTCF motifs at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs “facing” one another.Next, I will discuss the biophysical mechanism that underlies chromatin looping. Specifically, our data is consistent with the formation of loops by extrusion (Sanborn & Rao et al., PNAS, 2015). In fact, in many cases, the local structure of Hi‐C maps may be predicted in silico based on patterns of CTCF binding and an extrusion‐based model.Finally, I will show that by modifying CTCF motifs using CRISPR, we can reliably add, move, and delete loops and domains. Thus, it possible not only to “read” the genome's 3D architecture, but also to write it.