bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary Tryptophan‐Lactobacillus rhamnosus GG Interactions Modify Intestinal Differentiation and Function
Authors: Suntornsaratoon Panan , Flores Juan , Upadhyay Ravij , Lopez Paula , Patel Radha , Bandyopadhyay Sheila , Gao Nan , Ferraris Ronaldo
Published: May 2022

Although the influence of the gut microbiota on structure and proliferation of intestinal epithelial cells has been established, there is disconcordance on information related to cell type differentiation, because most studies on probiotics reflected an outcome from interactions among Lactobacillus, numerous existing microbes, and the host, with limited insight on the specific impact of Lactobacilli spp. For example, L. reuteri enhanced goblet and Paneth cell differentiation in intestine of conventionally‐raised chickens with an established commensal gut microbiota, confounding results. Here we fed gnotobiotic mice with tryptophan (t+) or trp‐free (t–) diets, followed by monocolonization with LGG (L+, ATCC 53103) or without (control, L–) for a total of 4 groups (L+t+, L+t–, L–t+, L–,t–), then ileum transcriptome was analyzed 3 wk later. L– mice remained germ‐free throughout while only LGG was associated with L+. Principal component analysis shows L+ or t+ by itself to modestly cluster away from L–t–, but the cluster depicting the combination of L+t+ was far removed from the others. The heatmap of whole ileum transcriptome shows several distinct groups of genes affected by t+ or by L+ alone. However, the simultaneous impact of L+t+ dramatically upregulates numerous gene clusters while downregulating a few. Volcano plots and Venn diagrams comparing L–t– with L–t+, or L+t– with L–t– indicate ~2300 unique genes affected by L+ (LGG) or t+ (tryptophan) alone. Comparisons between L+t+ and L+t– (effect of t with L) or between L+t+ and L–t+ (effect of L with t) each impact >3800 genes, suggesting that synergistic interactions between L+ and t+ primarily underlie the probiotic impact ascribed to LGG and dietary t. Many of the genes upregulated by L+t+ have functions typical of mature enterocytes: tight junctions, brush border membrane transporters and enzymes, intestinal bile acid metabolism, lipid synthesis and fatty acid b‐oxidation. Many downregulated genes are antimicrobial and secreted peptides typical of differentiated Paneth and goblet cells. This latter result was recapitulated when reexamined in conventional mice perfused for 4 h with LGG, as expression of many defensins decreased by >10‐fold, suggesting not only that this probiotic’s effects can arise from both acute ex vivo and chronic in vivo interactions with the host, but also in hosts housing a complex microbiota. Remarkably, Notch ligands Dll1 and Dll4 typically expressed in Paneth cells as well as Yap1, Axin2 and Ctnnb1 are upregulated in L+t+ ileum, suggesting downstream upregulation of Hes1 and repression of Atoh1, leading to suppression of the secretory lineage. In conclusion, LGG monocolonization may lead to synthesis of t+‐derived metabolites by both host and bacteria, leading to an enhancement of absorptive enterocyte functions and suppression of those by goblet and Paneth cells.
CD68, CD86 and CD163 Expression Profile in Breast Cancer Molecular Subtypes
Authors: Talaat Iman M. , Elemam Noha M. , Abdullah Hanan W.
Published: May 2022

Background and aim

Breast cancer (BC) is the second most common global cause of cancer deaths among women. Several immune cells are identified in the tumor microenvironment of BC patients, including tumor‐associated macrophages. We aimed at exploring the expression of distinct functional phenotypes using macrophages’ markers, where CD68 is a pan‐macrophage marker, CD86 is a marker expressed in polarized M1 subtype, and CD163 is expressed in M2 polarized subtype.

Methods

A retrospective study was performed using 90 formalin‐fixed paraffin‐embedded BC specimens for the immunohistochemical analysis of CD68, CD86 and CD163. Also, an in silico tool, UALCAN, was used on a larger cohort (n=1,081) of BC patients to investigate the expression of these markers. The macrophages’ markers were then associated with the clinicopathological parameters of BC patients. Triple‐negative BC cell line “CAL‐51” and luminal BC cell line “MCF‐7” were used to collect their respective supernatants that were added to THP‐1 derived macrophages. Then CD86 and CD163 were assessed using western blot.

Results

The pan‐marker of macrophages, CD68, along with the M1 CD86 marker and M2 CD163 marker, showed positive results in all the 90 investigated patients (Figure 1). CD86 expression was significantly associated with body mass index (BMI) and Ki‐67 proliferation marker. On the other hand, CD163 was significantly correlated with tumor size, estrogen, progesterone receptors, and BC molecular subtypes. Moreover, the high expression of CD86 and CD163 showed unfavorable outcome and survival of BC patients. In silicoanalysis revealed a significant increase in the CD86 expression in BC patients, especially in the triple‐negative subtype. Similarly, CD163 expression was found to be higher in the triple‐negative subgroup compared to the luminal group (Figure 2A). Additionally, in vitro work showed that the macrophages induced from the monocytic THP‐1 cell line express high levels of CD163 upon the exposure to conditioned media collected from the luminal BC cell line “MCF‐7”, thus showing M2 phenotype. On the contrary, CD163 expression was reduced upon the exposure to the conditioned media collected from the triple‐negative BC cell line “CAL51”, indicating more M1 phenotype (Figure 2B).

Conclusion

Tumor‐associated macrophages are associated with cancer progression and molecular subtypes. Hence, identifying the macrophages polarization profiles in each molecular subtype might aid their use as potential therapeutic targets.
Aspects of Mannheimia haemolytica cryopreservation
Authors: Nikitin Georgy , Makavchik Svetlana , Sukhinin Alexander , Smirnova Lubov
Published: May 2022

Mannheimia haemolytica causes severe pneumonia and septicemia in ruminant animals. The study aims to create an effective protective media for the long‐term storage of microbial cultures of M. haemolytica in a veterinary laboratory. The protective nutrient media were based on Hottinger's nutrient broth. Glycerol and ethylene glycol were used as cryoprotectants. Two protective media were obtained. Medium #1 consisted of nutrient broth (90%) and glycerin (10%), and medium #2 had nutrient broth (90%) and ethylene glycol (10%) in it. A homogeneous suspension 1.0 McFarland units turbidity was prepared from a pure culture of microorganisms (3.0 x 108 CFU/ml). The suspension was added to ampoules with an appropriate protective medium. The inoculated media were placed in a cryogenic storage dewar in liquid nitrogen at ‐196 ° C. The viability of M. haemolytica was determined by inoculation on blood agar and counting the number of living bacteria. The initial inoculum concentration of M. haemolytica was 3.0x107 CFU/ml. After 18‐20 hours of cultivation, it becomes 7.0x109 CFU/ml. The survival and safety of bacteria depend on the cryoprotectant. The results of the experiment showed that 10% glycerol provides 98.6% survival of M. haemolytica. The number of viable cells after cryopreservation was 6.9x109 CFU/ml. Ethylene glycol showed a 95.7% survival rate. The number of viable cells after cryopreservation was 6.7x109 CFU/ml. Without cryoprotectant, M. haemolytica showed a 40.0% survival rate with only 2.8x109 CFU/ml viable cells after defrosting. M. haemolytica causes turbidity and sediment formation at the bottom of the tube on Hottinger broth. After growing on semi‐liquid media (semi‐liquid agar with the addition of glucose or serum), bacteria show bipolar staining in smears. On Hottinger broth, with the addition of 10% defibrinated ram blood M. haemolytica formed small dewy colonies with an even edge without a hemolysis zone. The final species identification was performed by the proteomic method using MALDI‐TOF‐SM. All used media keep microorganisms' cultures in a stable, viable state. Thus, they can be successfully used for the storage of bacteria at low‐temperature conditions.
The Effect of Aerobic Exercise Training on Sympathetic Nervous Activity and Vascular Compliance in Patients with Chronic Kidney Disease
Authors: Jeong Jin Hee , Sprick Justin D. , DaCosta Dana A. , Mammino Kevin , Nocera Joe , Park Jeanie
Published: May 2022

Intro

Chronic kidney disease (CKD) is characterized by sympathetic nervous system (SNS) overactivity, which increases risk of cardiovascular (CV) disease and mortality. SNS overactivity can elevate CV risk by impairing vascular compliance and promoting inflammation. Aerobic exercise training exerts CV protective effects and has shown to reduce resting SNS level in populations at heightened CV risk. We tested the hypothesis that aerobic exercise training would reduce resting SNS activity and vascular inflammation and improve arterial compliance in patients with CKD.

Method

In this randomized controlled trial, sedentary individuals with CKD stages III‐IV underwent 12 weeks of aerobic cycling exercise (EX, spinning at 85% of maximal heart rate reserve, n=22) or stretching (ST, n=20, active control intervention). Both interventions were performed 45 minutes/session 3 days/week and matched for duration. Resting muscle sympathetic nervous activity (MSNA) was measured via microneurography, augmentation index (Aix75) was measured by brachial tonometry and vascular cell adhesion protein1 (VCAM) and tumor necrosis factor‐α (TNF‐α) were measured from venous blood samples. All measurements were performed at baseline and following the 12‐week intervention.

Results

There was a significant group by time interaction in MSNA (p=0.030) with no change in the EX group (32.8 ± 11.9 to 28.1 ± 12.9 bursts/min; p=0.215, n=15), but an increase in the ST group (30.2 ± 11.3 to 37.2 ± 12.9 bursts/min; p=0.042, n=10) after 12 weeks. The similar trend was observed in Aix75 (p=0.031) with no change in EX group (24.9 ± 7.9 to 24.3 ± 9.7; p=0.683, n=22), but an increase in ST group (22.6 ± 7.4 to 26.7 ± 10.5; p=0.012, n=20). We observed no significant change in VCAM and TNF‐α after 12 weeks in either group. The magnitude of change in MSNA was inversely associated with baseline MSNA in EX group (r=‐0.51, p=0.051). In the combined groups, MSNA was positively associated with Aix75 (r=0.38, p=0.06), VCAM (r=0.49, p=0.03) and TNFa (r=0.32, p=0.18) at baseline.

Conclusion

Our data demonstrates that 12 weeks of cycling exercise has beneficial effects on resting SNS activity and arterial compliance in CKD patients. Specifically, an increase in MSNA and Aix75 observed in the control group over 12 weeks was blunted following exercise training. This sympathoinhibitory effect of exercise training was independent of inflammation and showed greater magnitude in CKD patients with higher resting MSNA.
Ultrastructure of the mammary gland in lactating Saanen goats
Authors: Melnikov Sergey , Shchipakin Mikhail , Zelenevskiy Nikolay , Bylinskaya Darya , Khvatov Victor , Glushonok Sofia
Published: May 2022

The secretory cells of the mammary gland are very diverse in their size, shape, and structure. These are highly specialized cells adapted to the synthesis, accumulation, storage, and excretion of milk. The main feature of glandular cells is the presence of secreted substances. Electron microscopy shows that substances of different secretory cells types have various electron densities. Occasionally, two or more types of secretory granules differing in size and composition can be found in one cell. The material for the morphological study was small (2‐4 mm) tissue samples of goat lactating mammary glands. Pieces were taken from deeper areas of the breast parenchyma. Ultrathin sections with a 50‐70 nm thickness were obtained for electron microscopic analysis using Leica UC7 ultramicrotome. Electron microscopy was performed using a JEOL JEM 1011 microscope. Micrographs were obtained using a Morada camera (Digital Imaging Solutions Inc.). The secretion of the mammary gland occurs according to the apocrine type. Picture of the lactocytes' cytoplasm apical part separation into the lumen of the alveoli is often observed on semi‐thin sections. Thus, numerous droplets of material secreted by lactocytes are found in the lumen of the alveoli. The shape of lactocytes in the lactating mammary gland is close to cubic. The height of the epithelial layer, generally, is 10‐12 microns (µm). The milk ducts, like the alveoli, are dilated. Most of them contain a significant amount of osmiophilic material secreted by the cells of the mammary alveoli. The cells of the epithelium of the single‐layered ducts have a flattened or cuboidal shape. The diameter of the ducts varies noticeably depending on location in the gland parenchyma. The intralobular milk ducts are 10‐20 µm in diameter. At the same time, the larger interlobular ducts reach a diameter of 40‐50 microns and are located in groups in the connective tissue stroma of the mammary gland. Notably, an increase in the mammary alveoli mass in the lactating gland is associated with a decrease in connective tissue volume and the almost complete disappearance of fat cells groups from it. The milk ducts of the teat zone in the lactating gland are strongly expanded, round, or slightly oval‐shaped in cross‐section with a diameter of 30‐60 microns. In contrast to the deeper zones of the mammary gland, we were unable to detect secreted substances in the dilated milk ducts near the teat area. The milk ducts we studied are located close to the most terminal sections of the mammary gland excretory system (gland cistern and teat canal). The milk is inevitably washed out from the ducts during chemical fixation and subsequent stages of objects preparation for histological analysis.
Fetomaternal Cardiovascular and Renal Adverse Outcomes of Recurrent Preeclampsia in Rats
Authors: Wedn Abdalla M. , El‐Gowilly Sahar M. , El‐Mas Mahmoud M.
Published: May 2022

Preeclampsia (PE) is a serious pregnancy‐related disorder associated with considerable maternal and neonatal morbidity. Preeclamptic women have increased risk of PE recurrency during subsequent pregnancies. Clinically, recurrent PE is accompanied by more frequent maternal and fetal complications compared with the initial PE episode. Here, we investigated the impact of recurrent PE episode on the cardiovascular and renal profiles of female rats and their offspring. PE was induced by oral L‐NAME administration (50 mg/kg/day, gestation days 14‐21) for either single pregnancy or two consecutive pregnancies. Maternal and fetal abnormalities were compared with corresponding pregnancy controls. Recurrent and single PE caused similar increases in systolic blood pressure of mothers over last week of pregnancy and decreases in average weight of pubs. However, recurrent PE was associated with new onset elevation in renovascular reactivity to vasoconstrictors (phenylephrine and angiotensin II) compared with normal renal vasoconstriction in single PE dams. By contrast, the single, but not recurrent, PE was coupled with renal hypo‐reactivity to acetylcholine vasodilations. Examination of male and female progenies of single PE mothers revealed significant rises in blood pressure and renal vasodilatory dysfunction in female, but not male, offspring. Nevertheless, the attenuation in renovascular responsiveness to phenylephrine and angiotensin II was noted in both genders. Collectively, these data indicate that recurrent PE is associated with alleviation of single PE renal vasodilatory impairment as well as new onset hyperreactivity to vasoconstrictors. The results also highlight sexual dimorphism in the impact of PE on cardiovascular and renal profiles of the progeny. Future studies will be conducted to assess whether such sex‐related differences persist in the offspring of recurrent PE mothers.
In Vitro Screening Identifies TRPV4 and PAR1 as Targets for Endothelial Barrier Stabilization in COVID‐19
Authors: Michalick Laura , Mandzimba‐Maloko Bertina , Hamedi Shima , Dohmen Melanie , Brack Markus C. , Schulz Sabrina , Behrens Felix , Simmons Szandor , Müller‐Redetzky Holger , Suttorp Norbert , Kurth Florian , Corman Victor M. , Hocke Andreas C. , Witzenrath Martin , Hippenstiel Stefan , Kuebler Wolfgang M.
Published: May 2022

Study objective

Endothelial dysfunction and increased microvascular permeability are hallmarks of severe COVID‐19. At present, the underlying mechanisms of endothelial barrier failure in COVID‑19 remain elusive. Here, we show that increased thrombin activity in plasma from severe COVID‐19 patients activates endothelial protease‐activated receptor (PAR1), which mediates barrier failure by triggering TRPV4‐mediated Ca2+ influx in lung microvascular endothelial cells.

Methods

Citrate plasma was sampled as part of the Pa‐COVID‐19 cohort study (ethics approval EA2/066/20) from patients with severe COVID‐19 (high flow O2 or mechanically ventilated; WHO severity score: 5‐7) COVID‑19. Plasma samples were diluted to 10% (v/v) in cell culture medium without FCS and tested for their ability to disrupt barrier integrity of primary human pulmonary microvascular endothelial cells (HPMEC) monolayers by electrical cell‐substrate impedance sensing (ECIS), immunofluorescence for endothelial VE‐cadherin and F‐actin, western blot analyses of PAR‐1 cleavage, and real‐time Ca2+ imaging. Plasma from healthy donors served as control.

Results

COVID‐19 plasma had elevated thrombin activity while levels of antithrombin III, a key anti‐coagulant with thromboprotective function were decreased. COVID‐19 plasma caused endothelial barrier dysfunction as measured by ECIS and gap formation in HPMEC monolayers. Endothelial barrier disruption and endothelial Ca2+ influx in response to COVID‐19 plasma could be blocked by selective antagonists targeting thrombin (Argatroban), its receptor PAR1 (SCH79797), or TRPV4 (HC‐067047).

Conclusion

Here, we identify a novel signaling axis involving thrombin, its receptor PAR1, and TRPV4 as mechanism for increased microvascular permeability in COVID‑19. Targeting this signaling axis in endothelial barrier failure may provide a promising adjunctive therapy in COVID‐19.
Effects of Electrical Spinal Cord Stimulation on the Activity of the Hypothalamic‐Pituitary‐Adrenocortical System and the Sensitivity of the Gastric Mucosa to Ulcerogenic Stimuli
Authors: Filaretova Ludmila , Sakhno Denis , Moshonkina Tatiana , Gerasimenko Yury
Published: May 2022

Our previous results of two areas of unrelated researches are the basis of the present study. We demonstrated previously that the hypothalamic‐pituitary‐adrenocortical (HPA) axis is gastroprotective component of the brain‐gut axis and stress‐induced glucocorticoids are gastroprotective hormones but not ulcerogenic ones (Filaretova et al., 2016). At the same time recently electrical spinal cord stimulation began to be used for both experimental studies of motor functions regulation and rehabilitation of motor functions in patients with spinal cord injury (Gerasimenko et al., 2021; Moshonkina et al., 2016). The spinal cord stimulation directed to the activation of spinal locomotor related networks affected visceral systems as well. The aim of the present experimental work was to verify whether electrical spinal cord stimulation may activate the HPA axis and affect a sensitivity of the gastric mucosa to ulcerogenic stimuli in rats. Two ulcerogenic stimuli were used in anesthetized rats: 1) prolonged gastric ischemia/reperfusion (I/R, 30 min occlusion of celiac artery followed by 3 h of reperfusion); 2) indomethacin administration (IM, 35 mg/kg, sc). For spinal stimulation, stainless steel wire electrodes (AS632; Cooner Wire, California, USA) were fixed in fasciae between T11‐12 and L1–L2 vertebrae of anesthetized animals. Electrodes for recording motor responses to stimulation placed to the muscles of the hind limbs. A‐M System, Model 2100 stimulator was used for spinal cord stimulation. At the beginning motor thresholds determined by stimulation with monopolar rectangular pulses (1 ms). Then the spinal cord was stimulated with a frequency 30 Hz and a subthreshold current (350–700 μA) in order to exclude the influence of movements for 20 min. Sham‐stimulated (control) rats were subjected to the same manipulations except for the stimulation itself. The concentration of corticosterone in blood plasma was determined using commercial ELISA kits. The spinal cord stimulation resulted in an increase of blood glucocorticoid level 1 h later suggesting an activation of the HPA axis. The spinal stimulation, applied 1 h before the onset of ulcerogenic stimuli, also significantly attenuated gastric erosion formation induced by I/R and IM as well. The data suggest that the electrical spinal cord stimulation may activate the HPA axis and affect a sensitivity of the gastric mucosa to ulcerogenic stimuli in rats producing gastroprotective effect. We consider the results as a basis for further research to verify a possibility of an involvement of glucocorticoids in the mechanisms that may provide the beneficial effects of spinal neuromodulation.
Determining the End Nucleotide of Telomeres in Aspergillus nidulans
Authors: Day Jessica , Yimenu Meklit , Witte Graeme , Akins Allison , Kirk Karen
Published: May 2022

Telomeres are noncoding, repetitive DNA sequences at the ends of chromosomes that prevent DNA loss during replication. Telomerase is the ribonucleoprotein that extends telomeres by adding on telomere repeats. To do so, telomerase must recognize and bind to a specific sequence in the telomere repeats, which is often uncharacterized. In humans and our model system, Aspergillus nidulans, the telomere repeat is 5’‐TTAGGG‐3′. However, it is difficult to predict the order these nucleotides appear in the final telomere repeat because of end‐processing mechanisms that can occur at the chromosome ends. Previous work in Euplotes crassus and Tetrahymena thermophila has shown that their telomeres preferentially end 5’‐GGTTTTGG‐3′ and 5’‐TGGGGT‐’3 respectively. In humans however, the end nucleotide is much more varied on the G strand. It is currently unknown what the end nucleotide is in Aspergillus nidulans. To investigate this question, we employ a combination of telomere anchored PCR and quantitative PCR (qPCR) to preferentially amplify telomeres with specific endings. Initial results suggested all endings were present in genomic DNA, but to verify these results a control system was necessary. We have developed an oligonucleotide system consisting of synthetic telomere templates to test the validity of our method. With this control, we have observed that our system preferentially amplifies matching template and primer combinations, although there is some non‐specific binding between noncomplementary pairs. Thus, we have found suggestive preliminary evidence that we can use this system to assist in characterizing the end nucleotide of Aspergillus nidulans.
Development of a Vitamin A Deficient (VAD) Songbird
Authors: Olson Christopher R. , James Cadecia
Published: May 2022

Vitamin A deficiency (VAD) is a global health problem with effects on brain function through altered retinoic acid signaling. However, the specific effect of VAD on speech and vocal‐motor learning has not been studied. Songbirds possess a cortical brain circuit for vocal learning that relies on retinoic acid signaling during the critical period of vocal learning. To study VAD in a vocal learner we developed a special diet from white rice and sunflower seed, two grains that are naturally low in vitamin A and carotenoids, with other essential vitamins and minerals added back. This diet was fed to zebra finch family groups during chick rearing to evaluate the ability of parents to feed the diet to their chicks and the effects of a VAD diet on serum and brain retinol, chick growth and development. We found that adult birds readily consumed the rice/sunflower seed diet and fed it to their nestlings. Adults that consumed the diet maintained the same caloric intakes compared to when fed a regular seed diet and although these birds showed some decline in body mass, it remained within a normal range. Surprisingly, we detected a 28% decline in serum retinol at three weeks on the VAD diet in adult finches, a response that makes songbirds a highly useful model to study VAD. Juveniles that were fed the diet by the adults beginning at hatch also were able to consume and thrive. Compared to controls, chicks on the VAD diet showed no effects on growth rate between 10‐65 days, but the chick survival rate was 63% lower than that of controls with no chicks surviving beyond 65 days old. Chicks that were on the VAD diet did not exhibit sub‐song behavior between 35‐50 days (72 total hours of monitoring), whereas chicks that were on a seed diet or a rice/sunflower seed control diet (with retinol added back) showed song development. We also note that VAD affected eye shape and appearance, consistent with what is found during VAD in non‐avian models. Serum retinol of chicks on the VAD diet showed a 57% decline compared to controls. Further development of this model will require titration of the VAD to moderate levels that allow for song development and high chick survival. This study lays the groundwork for the use of modified diets in the songbird model to examine how VAD or other micronutrients may modulate neural development in the vocal circuit of a vocal learner, as well as open the door for the use of the model in high‐fat diet nutrition experiments and lipid transport studies.
Spatial Transcriptomics Reveal Potential Sex Differences in Gene Expression of the Supraoptic Nucleus
Authors: Nguyen Dianna H‐T , Phillips Nicole , Cunningham J. T.
Published: May 2022

The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory cells that play a key role in the regulation of fluid and electrolyte homeostasis. Although there are many well‐known sexually dimorphic regions of the hypothalamus, little is known about whether this is also true for the SON and whether there are sex differences in SON gene expression. Our study aims to address this knowledge gap by leveraging spatially‐resolved transcriptomics to better visualize gene expression profiles of cells in the SON of male and female rats and gain insight on their physiological functions without sacrificing morphological context. Visium Spatial Gene Expression (10x Genomics) was used to obtain spatially‐resolved gene expression data for the SON of adult male (n=3) and female (n=3) Sprague‐Dawley rats. Briefly, each brain was sectioned at 10μm thickness to collect coronal sections (~4x4mm) containing the SON and other brain structures. Each section was then mounted on the capture areas of Visium slides containing probes that bind mRNA. Next, the sections underwent the workflow as follows: 1) sample staining and imaging, 2) cDNA library preparation, 3) sequencing, and 4) analysis/data visualization. Data were analyzed using 10x Genomics’ Space Ranger and Loupe Browser applications to overlay spatial gene expression data with reference images. Results show that gene cluster analysis successfully differentiated myelinated fiber tracts from nuclei and identified several distinct neuronal populations in the coronal brain sections from both male and female rats. From the list of significant genes after performing differential expression analysis on the SON region using 10x Genomics’ Loup Browser, 74/157 genes were unique to the males, 228/311 genes were unique to the females, and 83 genes (e.g., Avp and Oxt) were common to both sexes. Preliminary Gene Ontology (GO) Enrichment and pathway analyses revealed GO terms and pathways related to: 1) homeostatic processes, regulation of peptide hormone secretion, and ion transport for the common genes; 2) ribosomes and translation for the significant genes unique to males; and 3) neurotransmitter release cycle and synaptic transmission for the significant genes unique to females, as some examples. These spatially‐resolved transcriptomic data suggest potential sex differences in SON gene expression that may be associated with processes and pathways important for osmoregulation and fluid balance. Future spatial transcriptomic studies will investigate changes in SON gene expression that contribute to sex differences in cellular mechanisms involved in body fluid homeostasis and possibly pathophysiology.
Spt5‐nucleic acid interactions are directly involved in promoter proximal pausing of RNA polymerase II
Authors: Dollinger Roberta , Gilmour David S.
Published: May 2022

Promoter proximal pausing of RNA polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires DSIF and NELF. DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. We use Drosophila melanogasteras a model system to address this question. We studied DSIF‐nucleic acid contacts made by the Spt5 NGN and KOW1 domains, which form a part of the upstream DNA exit tunnel, and the Spt5 KOW4 domain, which, along with the KOW5 domain, forms a clamp around the nascent RNA. Biochemical and structural studies have implicated these domains in pausing. We hypothesize that electrostatic interactions between Spt5 and the nucleic acid scaffold contribute to pausing by inhibiting the translocation of Pol II. We disrupted these electrostatic interactions by reversing the charge of key basic residues in Spt5 and used an electrophoretic mobility shift assay to measure Pol II binding and NELF recruitment. We then tested the pausing activity of the DSIF mutants in Drosophilanuclear extract depleted of wild‐type DSIF. Reversing the charge of six basic residues in the KOW1 domain resulted in weaker binding to Pol II. Though this mutant was still able to recruit NELF, it showed greatly reduced pausing activity. Reversing the charge of four basic residues in the KOW4 domain only modestly reduced binding to Pol II, had no impact on NELF recruitment, and significantly impaired DSIF’s pausing function. Thus, nucleic acid contacts made by the KOW1 domain of Spt5 promote pausing by mediating Pol II‐DSIF interactions (and therefore NELF recruitment) while the KOW4 domain interactions with the nascent transcript contribute to pausing directly. We also reversed the charge of two basic residues in the NGN domain and found that these changes had no effect on DSIF binding and NELF recruitment but did result in a modest decrease in pausing activity. One of these basic residues is an arginine located in an alpha helix sequence that is conserved in eukaryotes with NELF and promoter proximal pausing but is absent in eukaryotes lacking NELF. To test whether this alpha helix is required for promoter proximal pausing, we replaced the Drosophila helix sequence with unstructured loop sequences from Komagataella pastoris, Saccharomyces cerevisiae, and Caenorhabditis elegans, none of which have NELF. These mutants were able to bind Pol II and recruit NELF but exhibited significantly reduced pausing activity, suggesting that a short alpha helix in the NGN domain is critical for stabilizing the promoter proximal pause. This is likely achieved by optimally positioning a conserved arginine to interact with the DNA scaffold.
Direct evidence of a sympathetically‐mediated cardio‐inotropic response to baroreceptor unloading in rats
Authors: Stewart Liam , Wainman Liisa , Ahmadian Mehdi , Duffy Jennifer , Seethaler Rudi , Mueller Patrick J. , Eves Neil , West Christopher R.
Published: May 2022

Rationale

The baroreflex is a cardio‐autonomic reflex that is responsible for buffering acute oscillations in blood pressure. Contemporary evidence suggests that, when stimulated, baroreceptors activate a neural reflex arc via two efferent pathways: the vascular‐sympathetic arm to control peripheral resistance, and the cardio‐vagal arm to control heart rate. However, since sympathetic post‐ganglionic fibres innervate both the vasculature and left ventricle (LV) it follows that the heart may also contribute to hemodynamic regulation during baroreceptor unloading through alterations in cardiac inotropy, yet to our knowledge this has not been empirically tested using a load‐independent index of LV inotropy.

Aim

1) To determine whether there is an increase in LV inotropy in response to baroreceptor unloading, and 2) to parse out whether this response occurs via sympathetic activation, parasympathetic withdrawal, or a combination of the two.

Hypotheses

There will be an increase in LV inotropy in response to baroreceptor unloading, and this response will occur via activation of the sympathetic nervous system.

Methods

10 male Wistar rats were anesthetized (urethane) and instrumented with a LV pressure‐volume catheter to measure the maximal rate of pressure generation for a given end‐diastolic volume (dP/dtmax–EDV mmHg·s‐1·µl‐1) as a load‐independent metric of cardiac inotropic function. Each rat was then placed in a customized servo‐controlled lower‐body negative pressure (LBNP) chamber that was programmed to reduce mean arterial pressure (MAP) by 10% for 60s to mechanically unload baroreceptors. The protocol was repeated in the same animal following infusions of lactated ringers (control), esmolol (β1 adrenergic receptor antagonist; blocks cardiac sympathetic transmission), atropine (muscarinic receptor antagonist; blocks cardiac parasympathetic transmission), and esmolol+atropine (full cardiac autonomic blockade). Between‐condition differences in LV inotropy were assessed using mixed‐models with Bonferroni corrected post‐hoc pairwise comparisons.

Results

Under control conditions, dP/dtmax–EDV increased significantly (25.8±4.9 vs 29.9±6.7 mmHg·s‐1·µl‐1; p=0.002) during unloading. Sympathetic and full cardiac blockade significantly reduced baseline dP/dtmax–EDV (10.8±2.1 and 13.5±3.0 mmHg·s‐1·µl‐1, respectively; p<0.001 for both), and abolished the response to baroreceptor unloading (10.6±2.1 and 14.3±4.0 mmHg·s‐1·µl‐1, respectively; p=0.999 for both). Parasympathetic blockade had no effect on baseline dP/dtmax–EDV (26.1±5.5 mmHg·s‐1·µl‐1; p=0.999) and the increase in dP/dtmax–EDV during baroreceptor unloading was maintained (30.5±8.3 mmHg·s‐1·µl‐1; p<0.001).

Conclusion

These results provide direct evidence of a sympathetically‐mediated increase in cardiac inotropic function in response to baroreceptor unloading in rats.
Effect of Social Isolation on Ovarian Follicles in Female Mice
Authors: Rice Nathan E. , Pack Adam , Provost Terri , Keesom Sarah
Published: May 2022

Stay‐at‐home orders and social distancing have been central to the public health strategy employed during the COVID‐19 pandemic. While these measures are essential for reducing the transmission of COVID‐19, surveys administered during the pandemic revealed that this decrease in social interaction also resulted in increased loneliness and isolation in human populations over the past year. Social interaction is an important component of overall human wellness. In fact, social contact and social support are associated with better health and decreased mortality in humans. More studies are needed to specifically investigate how social isolation affects reproductive systems. Based on the rationale that environmental stimuli can influence hormonal cascades and therefore affect reproduction, we hypothesized that the lack of social stimuli experienced during social isolation influences the production of ovarian follicles. To test this, three‐week‐old female Swiss‐Webster mice were placed in individual housing or in groups of three for five weeks, then their ovaries were harvested and embedded in Durcupan. From each right ovary, we cut 110 sections at 2‐μm thickness, 20 sections at 10‐μm thickness, and 100 sections at 2‐μm thickness. The cuts were oriented transversely to the oviduct. An observer, blind to experimental treatment, evaluated sample sections taken every 50 µm by counting the total number of follicles present. In our preliminary analysis, we observed fewer follicles in individually housed mice than in group‐housed mice. These preliminary results suggest that social isolation has implications for reproductive health and, in a larger context, demonstrate the importance of intraspecies interactions for social animals.
Characterization of CARM1 Inhibition in Skeletal Muscle
Authors: Webb Erin K. , Ng Sean Y. , Stouth Derek W. , van Lieshout Tiffany L. , Mikhail Andrew I. , Ljubicic Vladimir
Published: May 2022

Coactivator‐associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of arginine residues of target proteins critical for health and disease. CARM1 has recently emerged as an important regulator of skeletal muscle biology. The purpose of this study was to further investigate CARM1 biology in skeletal muscle. For our first experiment, we utilized 3‐month‐old male and female mice (n = 3/sex) that were housed and cared for in accordance with Canadian Council for Animal Care guidelines. Mice were treated with either a CARM1 inhibitor (150 mg/kg EZM2302; Epizyme, Inc.) or vehicle via oral gavage BID for 2, 4, or 8 days. Tissues were collected 6 hours following the final dose and muscle mass was recorded. Western blotting was performed to evaluate protein expression in the tibialis anterior (TA) muscle and liver. To assess CARM1 activity, we investigated the arginine methylation status (i.e., the methylated form of the protein relative to its total amount) of known CARM1 substrates BAF155 and PABP1, as well as the myocellular level of arginine methylated proteins preferentially marked by CARM1. We observed similar outcomes between males and females, so the data were collapsed by sex (n = 6). CARM1 inhibition significantly reduced BAF155 methylation status in the TA muscle by 70‐80% between 2‐8 days of treatment. Muscle PABP1 methylation status was also attenuated (p < 0.05) by 60‐65% after 4 and 8 days of exposure. Similarly, arginine methylation of CARM1‐specific substrates across all muscle proteins was reduced by ~50% (p < 0.05) between 2‐8 days of CARM1 inhibitor administration. The attenuation of BAF155 and PABP1 methylation status, as well as arginine methylated CARM1‐specific substrates, was greater in liver as compared to muscle. Next, we completed additional experiments to assess the effect of pharmacological CARM1 inhibition on skeletal muscle function. After 11 days of CARM1 inhibitor treatment, we observed a sex‐specific reduction (p < 0.05) in exercise capacity and muscular endurance such that muscle function in female mice was unaffected by CARM1 suppression. Collectively, these results suggest that global, pharmacological CARM1 inhibition preferentially impacts liver enzyme activity over muscle. Furthermore, CARM1 inhibitor treatment may affect muscle performance with males being more sensitive to the functional outcomes of CARM1 suppression than females. Future work in our laboratory will examine the mechanisms and functional consequences of sex‐based differences in CARM1 biology within skeletal muscle.
Anteriorly Rotated Liver Anomaly in a Prosection Cadaver
Authors: Hurley Mary
Published: May 2022

The abdominal contents of a 85 year old male cadaver whose cause of death was decompensated congestive heart failure presented with an anteriorly rotated liver that had an enlarged right lobe and much reduced left lobe. The caudate lobe was also much reduced. The gall bladder was oriented vertically, facing the anterior abdominal wall. Other visceral abnormalities included the stomach, which was horizontal in orientation, with the greater curvature facing the anterior abdominal wall and greater omentum shifted to the left. The ascending colon continued beyond the hepatic flexure, and the transverse colon was located in the left upper quadrant. The descending colon was shifted towards the right and the sigmoid colon was enlarged, and it and the sigmoid mesocolon were located in the right lower quadrant. The vasculature of the viscera originated from the unpaired abdominal aortic branches in typical fashion, except that some were of different lengths to reach the organs that they supplied. One such vessel was the proper hepatic artery as it had to traverse the abdominal cavity further horizontally than typical due to the displacement of the liver. The inferior mesenteric artery took a looping path to the right lower quadrant due to the displacement of the sigmoid colon. The middle colic artery branch of the superior mesenteric traveled vertically and to the left to supply the transverse colon located in the left upper quadrant.

Literature provides few examples of this anomaly. An autopsy of a 39 year old African American female had similar presentation of the liver and gall bladder but no report of unusual presentation of the colon.(https://www.hindawi.com/journals/crihep/2013/493713/(2013)) It was also reported as a result of a CT scan of an 82 year old Indian male that was performed due to left flank pain (
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159280/(2017)). Noted during exploratory surgery of a 30‐year‐old African American male with a pleuroperitoneal hiatus that also had congenital deformities of the right upper extremity and a club foot, the liver was rotated into the left upper quadrant with a posteriorly placed gall bladder (Diseases of the Chest 29: 583‐4(1956)).
Phenotypic Changes Produced by Endogenous DNA Damage in Yeast Mutants Deficient in Recombination and Base Excision Repair
Authors: Berry Armand M.
Published: May 2022

DNA is under constant attack by exogenous and endogenous factors. Endogenous sources of DNA lesions include free radicals such as reactive oxygen and nitrogen species (RONS), as well as many naturally occurring enzymatic and chemical processes. To prevent mutations and possibly cell death these lesions must be repaired prior to DNA replication. There are five major pathways that are utilized for repair depending on the type of DNA damage. Eukaryotes utilize two pathways, homologous recombination (HR) and nonhomologous end‐joining (NHEJ), for repair of DNA double‐strand breaks (DSBs). In addition, the cells utilize base excision repair (BER) for repair of damaged or missing bases. Previous work in the lab has shown that HR‐deficient yeast mutant cell cultures exhibit high levels of G2 phase cells during normal growth, without exposure to exogenous DNA damaging agents. Inactivation of DNA damage checkpoint genes, but not spindle checkpoint genes, abolished the high G2 cell phenotype, indicating that the damage checkpoint is persistently activated in the cells. In a screen of yeast mutant library strains deficient in each of the major repair pathways, the high G2 phenotype was observed in HR‐ and BER‐deficient cells, but not in mutants specifically defective in nucleotide excision repair (NER), mismatch repair (MMR) or NHEJ.

Experiments performed for the current project have assessed the consequences of inactivation of the HR and BER pathways on yeast cells in more detail. The high G2 cell phenotype was heightened in both diploid HR (rad52) and BER (apn1, ogg1) mutants relative to their haploid counterparts. Expression of DIN1 (RNR3), a DNA damage‐inducible gene, was found to be constitutively induced in the HR mutant cells. Preliminary tests of the role of free radicals in generation of endogenous signaling were performed by overexpression of Yap1p, a transcriptional activator of antioxidant enzyme genes. The high G2 cell phenotype was abolished in ogg1 mutants and strongly reduced in rad52 cells when Yap1p was overexpressed. Current work is focused on (a) analysis of DIN1 promoter activation in BER mutants, (b) assessment of the impact of neutralization of free radicals using chemical antioxidants, and (c) measurement of phosphorylated Rad53 checkpoint protein levels using Western blots to characterize the DNA damage checkpoint signaling response.
Significance of the Translation Initiation Machinery Recruiting Structures in the Blackcurrant Reversion Virus RNA 1
Authors: Lee Julie Y. , Zhou Liheng , Baquero‐Galvis Laura D. , Filbin Megan E.
Published: May 2022

Unlike most eukaryotic translation which is initiated by the 5′‐m7GpppN cap (5′cap), the two genomic RNAs (RNA 1 and 2) from the Blackcurrant Reversion virus (BRV) do not have a 5′ cap but rather has special region in 3′ untranslated region (UTR) called a cap‐independent translation enhancer (CITE). CITE RNA structures bind translation initiation machinery and delivers that machinery to the 5′ UTR, typically via an RNA kissing‐loop interaction. Our research focuses on the specific RNA structures called translation initiation machinery recruiting structures (TIMRS) in the BRV RNA 1 3′ CITE and 5′ UTR, and their significance in facilitating translation by delivering translation machinery to the 5′ end of the RNA. Using site‐directed mutagenesis to alter the TIMRS and a luciferase reporter to measure protein production of the mutants, we are analyzing the relationship between structure and function. Thus far we have found altering the TIMRS in the 3′ CITE structure does show a difference in protein production compared to the wild type, which indicates the structures' significance and their contribution in translation efficiency. We believe the TIMRS is responsible for recruiting translation machinery or possibly the ability to “kiss” to 5′ UTR stem loop. This study can aid in understanding not only plant viruses but other animal or human RNA viruses that utilize 3′ or 5′ structures to regulate cap‐independent translation.