bluuebunny/crossref_metadata_2025 huggingface dataset:
Possible effects of ASIC‐BK interaction on glial growth at normal and acidic pH
Authors: Guercio Jennifer C , Petroff Elena
Published: June 2021

Normal glia and glioma tissue express acid sensing ion channels (ASICs) and large conductance calcium and voltage‐activated potassium (BK) channels. At normal brain pH (7.4), ASICs interact with and inhibit BK channels, and this interaction is disrupted at an acidic pH (Petroff et al., PNAS 2008). Inhibition of BK at normal pH may inhibit growth of glial cells. At an acidic pH, this interaction is disrupted, and the active BK channels may foster cell proliferation, such as at a site of trauma or injury. This hypothesis was tested by growing isolated mouse glial cells from the cortex at normal pH (7.4) to simulate a normal brain environment, at a low pH of 7.0 present in tumors, and at both pH 7.4 and 7.0 in the presence of 200nM of the potassium channel blocker charybdotoxin. Our data demonstrate that cell proliferation is significantly increased at pH 7.0 as compared to normal pH conditions (7.4). The presence of charybdotoxin inhibited glial growth at both pH's. These results suggest the importance of extracellular pH and regulation of potassium channel activity for glial cell proliferation. This work is supported by the MSU startup funds for EP.
Golgin‐97 facilitates Golgi Export of Inward Rectifier Potassium Channel
Authors: TANEJA TARVINDER KAUR , Ma Donghui , Welling Paul A
Published: June 2021

The inward rectifying potassium channel, Kir2.1, is selected as cargo into trans‐Golgi export carriers through a unique signal‐dependent mechanism. Mutations in the signal or depletion of the trafficking machinery (AP‐1) that decodes it prevents export of the channel from the TGN. Surprisingly, they also cause marked accumulation of the channel throughout the entire Golgi apparatus, suggesting anterograde transport of Kir2.1 through the Golgi may be closely coupled to its export from the TGN. Here we explore the mechanism. A systematic screen of Golgi tethers indentified Golgin‐97 as a Kir2.1 binding partner. In vitro studies revealed the interaction is direct, occurring between the GRIP domain of Golgin‐97 and the cytoplasmic domain of Kir2.1. Imaging and interaction studies in cells revealed that Golgi‐97 binds to the channel en route through the Golgi. Knockdown of Golgin‐97 using siRNA prevented exit of Kir2.1 from the Golgi. These observations are consistent with the idea that Golgin‐97 directly interacts with Kir2.1 in the cis‐Golgi for selection into specialized exit sites of the TGN. This project was supported by funds from NIH and American Heart Association.
Effects of Hypoxia on Gene Expression in the Elderly
Authors: Toby Inimary , Burian Dennis , McNeely Eileen
Published: June 2021

Many patients with underlying pulmonary disease travel each year by air. These patients may be at higher risk for developing complications due to decreased oxygen tension within their lungs, or hypoxia. At high altitudes, the lower air pressure makes it more difficult for oxygen to enter our vascular systems. In addition, aviators who fly above 12,500 feet in an unpressurized aircraft without supplemental oxygen may be susceptible to the effects of hypoxia. It is unclear what determines an individual's response and subsequent compensation to hypoxia. We enrolled a total of 62 elderly subjects aged 50–80 from three health categories: 23 healthy, 18 cardiac‐compromised, and 21 smokers. Subjects participated in a simulated flight within a hypobaric chamber under both control and exposure conditions at an altitude of 7,000 ft. We utilized microarray analysis and quantitative PCR (qPCR) to access gene expression of the subjects in response to altitude exposure. Using microarray analysis, we discovered ~155 genes involved in the physiological response to altitude exposure. The main biological functions involved were cell‐to‐cell signaling, hematological system function, and tissue development. Of the 155 genes, we utilized qPCR to measure the levels of expression for the top 35 genes. We found that hypoxia led to alterations in gene expression of important factors in the pulmonary vasculature. Our data suggests that several biomarkers exist that might be predictive of an individual's response to hypoxia.

This work was supported by the Federal Aviation Administration, Office of Aviation Safety
The PPARγ Ligand, Rosiglitazone Attenuates Hypoxia‐Induced Endothelin Signaling in vitro and in vivo
Authors: Kang Bum‐Yong , Ritzenthaler Jeff D. , Hart Michael C.
Published: June 2021

PPARγ activation attenuated hypoxia‐induced pulmonary hypertension (PH) in mice although the mechanisms for these effects remain to be completely defined. We hypothesized that PPARγ attenuates hypoxia‐induced endothelin‐1 (ET‐1) signaling. Human pulmonary artery endothelial cells (HPAECs) were exposed to hypoxia (1% O2) or normoxia for 72 h and treated ± rosiglitazone (RSG, 10 μM) during the last 24 h. Male C57BL/6 mice were exposed to hypoxia (10% O2) or normoxia (21% O2) for 3 wk ± gavage with RSG (10 mg/kg/d) for the final 10 d of exposure. Selected HPAECs were treated with chetomin (HIF‐1α inhibitor, 25 nM) or with CAPE (NF‐κB inhibitor, 20 μM) for 3 h. HPAEC proliferation was measured with MTT assays. Our findings demonstrate that hypoxia increased ET‐1, endothelin converting enzyme‐1, endothelin receptor A and B levels in mouse lung and in HPAECs and increased HPAEC proliferation. Treatment with RSG attenuated these hypoxia‐induced increases. Hypoxia‐induced increases in HIF‐1α and NF‐κB nuclear binding were attenuated by RSG. Treatment with chetomin or CAPE also prevented hypoxia‐induced increases in ET‐1 mRNA levels. These findings suggest that PPARγ attenuates hypoxia‐induced ET‐1 signaling by inhibiting activation of hypoxia‐responsive transcription factors. Targeting PPARγ represents a novel therapeutic strategy to inhibit ET‐1 signaling that contributes to PH pathogenesis.
A molecular signature of resistance training‐induced human skeletal muscle hypertrophy
Authors: Thalacker‐Mercer Anna , Merritt Edward , Cui Xiangqin , Bamman Marcas
Published: June 2021

Using K‐means cluster analysis, we have previously shown that human adults have widely divergent myofiber growth responses to lower body progressive resistance training (PRT) with some having no response (NON), a modest response (1,111μm2 mean myofiber growth, MOD), or an extreme hypertrophic response (2,475μm2, XTR) PMID: 17395765. Underlying mechanisms for this differential growth response are largely unknown. The purpose of this study is therefore, to determine whether a molecular signature exists, prior to 16 weeks of PRT, which could predetermine the skeletal muscle hypertrophic response in adults. mRNA was isolated from muscle biopsies taken from the vastus lateralis of 44 previously clustered men and women (aged 20–75y). Agilent 4X44K single color genechips were used to determine differences in gene expression among the three clusters and identify informative genes which could predict the myofiber response phenotype to PRT. Microarray results were followed‐up with qRT‐PCR and immunoblotting. By comparing XTR to NON, our results suggest distinctive molecular signaling networks that may underlie the individual differences in potential for mechanically‐induced myofiber hypertrophy. Support: NIH 1R01 AG017896, VA Merit Review
Presence of evolutionary conserved gene expression signatures during progression of Barrett's esophagus to esophageal adenocarcinoma in human and rat
Authors: Kim Soo Mi , Lee Ju‐Seog
Published: June 2021

Esophageal adenocarcinoma (EAC) in Barrett's esophagus (BE) patients is thought to arise from a sequence of growth‐promoting genetic alterations due to chronic esophagitis. However the molecular events underlying the pathogenesis of BE and EAC remain largely unclear. To define the biological mechanisms of EAC development, we performed a cross‐species comparison of gene expression data from human and rat tissues, seeking the evolutionarily conserved gene expression patterns during tumor progression across both species. We used a non‐carcinogenic rat model that closely resembling the natural history of EAC development in human. Using Illumina beadarray platform, we first generated gene expression data from human tissues 28 NE, 72 BE, and 75 EAC as well as from rat tissues 15 NE, 18 BE, and 30 EAC. Statistical analyses on human and rat gene expression data revealed gene expression patterns significantly associated with progression of disease (NE to BE and BE to EAC). Using orthologous genes presented in both microarray platforms, we identified 397 genes are commonly altered as NE progresses to BE and 205 genes including SPP1 were altered as BE progresses to EAC in both species. Systems‐level characterization of gene expression data from human and rodent could become reliable biomarkers for early detection or therapeutic targets for treatment of EAC patients.
Isolation of single cells from murine late distal convoluted tubules and connecting tubules
Authors: Jensen Thomas Buus , Jensen Uffe Birk , Fenton Robert A , Praetorius Helle A , Praetorius Jeppe
Published: June 2021

Proteomic studies of late distal convoluted tubule (DCT2) and connecting tubule (CNT) cells have so far been hindered by technical difficulties in isolating these cells with sufficient yield and purity. We exploited a mouse model with endogenous expression of enhanced green fluorescent protein (EGFP) driven by the transient receptor potential vallinoid 5 (TRPV5) channel promoter. Whole kidneys were minced and incubated with 1 mg/ml proteinase K and 1 mg/ml collagenase II for 45 min at 37°C agitated at 180 RPM. The tubule‐suspension was treated with 0.12% trypsin in Ca2+‐free buffer for 2 × 5 min to obtain single cells. Cell separation and morphology were evaluated by light microscopy. Blood vessels and cell clusters were removed by passing the cell suspension through a 40‐μm mesh before fluorescence activated cell sorting. Cells were sorted on a FACS AriaIII (BD) with 100 μm nozzle at 20 psi and 488 nm excitation. Dead cells (approximately 2%) were excluded using propidium iodide. By this method, the yield corresponded to approximately 2.5×107 cells per mouse kidney of which 6×105 were GFP positive (2.4% of all cells) or 38 μg protein per kidney. Thus, we have developed a protocol that may allow proteomic analysis of mouse DCT2/CNT cells with virtually no contamination with cells from other renal structures or with intercalated cells from DCT2/CNT. Supported by the Danish Council for Independent Research | Medical Sciences.
Kv7 Channels in Airway Smooth Muscle Cells as Targets for Asthma Therapy
Authors: Kakad Priyanka Prakash , Brueggemann Lioubov I , Cribbs Leanne L , Byron Kenneth L
Published: June 2021

Asthma is commonly associated with airway hyperresponsiveness (AHR) involving hypercontraction of airway smooth muscle cells (ASMCs). The mechanisms underlying this process are, however, not well understood. Here we test the hypothesis that bronchoconstrictor agonists suppress Kv7 potassium currents leading to ASMC contraction. We detected Kv7.1–7.5 mRNAs in guinea pig lung by RT‐PCR and Kv7.3–7.5 proteins by immunostaining of isolated ASMCs. Function of Kv7 channels was demonstrated in freshly isolated guinea pig ASMCs: currents measured by perforated patch whole cell voltage clamp displayed electrophysiological characteristics (slow voltage‐dependent non‐inactivating Kv currents with a very negative activation threshold (~−60mV) and a V0.5 ~ −31 mV) and pharmacological characteristics (enhancement by selective activators, suppression by selective blockers) of Kv7 currents. Bronchoconstrictor agonists methacholine (100 nM) and histamine (30 μM), significantly suppressed Kv7 currents and this effect was reversed by Kv7 channel activators. Our results provide the first evidence that Kv7 channels are present in ASMCs, that their activity is suppressed by bronchoconstrictor agonists, and that this effect can be reversed by Kv7 channel activators. Our findings reveal novel mechanisms for ASMC hypercontraction and identify a potential new therapeutic strategy for the treatment of AHR in asthma.
AN AIRWAY ANTI‐INFLAMMATORY ROLE FOR FISH OIL SUPPLMENTATION
Authors: Ade Carl J , Rosenkranz Sara K , Harms Craig A
Published: June 2021

Environmental and dietary factors cause airway immune cells to produce mediators that increase tissue inflammation and smooth muscle constriction. A high‐fat meal is one stimulus that may increase airway inflammation in healthy individuals. Supplementation with omega‐3 fatty acids can have anti‐inflammatory properties via modification of arachidonic acid metabolism.

Aim

To determine if omega‐3 fatty acid supplementation via fish oil will mitigate the airway inflammatory response induced by a single high‐fat meal (HFM).

Methods

Non‐asthmatic males (22±3 yrs) were supplemented with 3 mg×day‐1 fish oil (FO; n=5) or a placebo (CON; n=6) for 3 weeks. Exhaled nitric oxide (eNO; a marker of airway inflammation), impulse oscillometry, pulmonary function, and triglycerides were measured prior to and 2 hours following a HFM.

Results

Following a HFM, triglycerides increased in both FO and CON groups (79% and 118% respectively, P<0.05). The increase in eNO was higher in the CON group compared to the FO group (26.6 ± 19.6% vs. −2.6 ± 13% respectively, P<0.05). FVC was decreased in the CON group (5.32 ± 0.77 pre‐HFM vs. 5.12 ± 0.76 l post‐HFM, P<0.05) but was unchanged in FO group (4.88 ± 0.91 pre‐HFM vs. 4.88 ± 0.80 l post‐HFM, P>0.05).

Conclusion

A HFM increases airway inflammation and decrease pulmonary function. Omega‐3 fatty acid supplementation via fish oil protects against HFM associated changes in airway health.
Cigarette smoke and A‐kinase anchoring proteins (AKAPs) in human airway smooth muscle function
Authors: Schmidt Martina , Oldenburger Anouk , Poppinga Wilfred , Roscioni Sara S , Heijink Irene H , Timens Wim , Skroblin Philip , Klussmann Enno , Maarsingh Harm
Published: June 2021

COPD is a chronic inflammatory disease mainly caused by cigarette smoke (CS) and characterized by infiltration of (activated) inflammatory cells. Airway smooth muscle (ASM) also contribute to inflammatory cytokine release, smooth muscle contraction, and airway remodeling. Notably, cAMP‐elevating β2‐agonists and phosphodiesterase inhibitors differentially diminish COPD symptoms. Compartmentalization of cAMP by AKAPs could explain distinct cAMP responses.

In human ASM, CS‐induced IL‐8 release was decreased by fenoterol, the PKA activator 6‐Bnz‐cAMP, while the Epac activator 8‐pCPT‐2′‐O‐Me‐cAMP showed only slight effects. Addition of PKA‐AKAP interaction inhibitor Ht31 augmented the IL‐8 release. RII overlay, western blots and RT‐qPCR demonstrated expression of AKAP79, AKAP250 and AKAP450. CSE decreased AKAP250, whereas AKAP79 was less affected and AKAP450 even enhanced, indicating disturbation of AKAP functions by CSE. Importantly, similar changes in AKAP expression were found in lung tissues of COPD patients. Our studies provide the first relation between cigarette smoke and the coordination of cAMP signaling by AKAPs. Supported by the Dutch Asthma Foundation and a Rosalind Franklin Fellowship.
Impairment of epithelial cell endocytosis by mistargeted SP‐C I73T mutant protein
Authors: Hawkins Arie Ellen , Zhao Ming , Beers Michael F. , Mulugeta Surafel
Published: June 2021

Interstitial lung disease in both children and adults has been linked to mutations in the lung‐ specific Surfactant Protein C (SP‐C) proprotein. Among these, the missense mutation, SP‐CI73T, accounts for more than 30% of all reported SP‐C mutations. We previously reported that in A549 and HEK293 cell lines, wild type SP‐C was directly targeted to lysosomal‐like vesicles, while SP‐CI73T was uncharacteristically routed to the plasma membrane, and subsequently sorted to the lysosomes via early endosomes. This mistargeting causes impaired cellular lipid uptake and degradation. To determine whether the diminution of lipid uptake is the product of a globally dysfunctional endocytic pathway, cellular uptake of dextran was examined in HEK293 cells stably expressing either the wild type or the mutant SP‐C isoforms. We found that internalization of dextran was significantly reduced in cells expressing SP‐CI73T compared to the wild type control. Thus, our data further establishes the functional impairment caused by the SP‐CI73T mutation, and supports our previously proposed cellular mechanism and paradigm for mistargeted proteins involved in the disruption of endosomal/lysosomal sorting machinery.

Supported By NIH HL090732 (SM), NIH HL19737 (MFB)
Novel tools for investigations of cytoskeletal re‐arrangement during alveolar epithelial‐mesenchymal transition
Authors: Ehrhardt Carsten , Medina Carlos , Davies Anthony Mitchell , Buckley Stephen Timothy
Published: June 2021

Inherent in epithelial‐mesenchymal transition (EMT) is the establishment of an elongated cell morphology and formation of stress fibres. The influence of these cellular alterations on the biomechanics of lung parenchyma remains unclear. Alveolar A549 cells, after exposure to TGF‐β1, were studied using atomic force microscopy (AFM) and high content analysis (HCA) imaging.

A549 cells were grown with or without TGF‐β1 in the presence or absence of a collagen I substrate for 72 h. Using AFM, the Young′s modulus of living cells was measured in contact mode. Surface membrane features were assessed on fixed cells using intermittent contact mode. In parallel, morphological and fluorescence staining intensity/distribution parameters were evaluated using a HCA imaging system.

TGF‐β1 resulted in a greater than two‐fold increase in cell stiffness. This effect was augmented in the presence of collagen I. TGF‐β1‐treated cells also exhibiting a rougher surface profile with notable protrusions. In accordance with these findings, concurrent quantitative examination of the morphological attributes of stimulated cells using HCA, revealed dramatic alterations in cell shape, F‐actin content and distribution.

These data indicate a strong correlation between the cytoskeletal‐associated cellular architecture and the mechanical dynamics of alveolar epithelial cells undergoing EMT.
A new foramen on ala of sacrum
Authors: Singh Rajani
Published: June 2021

A new foramen on posterior part of ala of sacrum was found during routine osteology class of MBBS students in the Department of Anatomy, CSM Medical University Lucknow, UP, India.

Almost no literature is available on this virgin foramen therefore, theory of this variant and clinical significance need to be conceptualised. So objective of this study has been set to add new model of anatomical theory and to develop clinical implication of this foramen.

Therefore, sixty six sacra were examined and analysed for incidence, position, shape and size of foramen Rajani (as named). Foramen is located at 8mm from sacral canal, 20mm from posterior tip of ala of sacrum and 5mm from posterior border of body of first sacral vertebra with incidence of 1.5%. It is oval shaped having its horizontal and vertical dimensions as 6 mm, 4mm. It communicates with the sacral foramina and sacral canal.

The structures, common iliac, or iliolumber artery or lumbosacral trunk is likely to pass through the foramen Rajani. These structures may be damaged during pedicle screw fixation or instrumentation by orthopaedic surgeons and neurologists.

Author could not avail the financial support from any institution.
A rare variant of the median nerve as a reminder of the patient uniqueness
Authors: Kumka Myroslava
Published: June 2021

Objective

Despite the wealth of data concerning anatomical variation of the median nerve, it remains important to continue describing rare variations as they arise. This study represents a unique variation of the median nerve in the human forearm.

Methods

32 upper limbs of 16 embalmed cadavers were utilized using anatomical dissections.

Results

The median nerve in a right forearm of a 61 year old male cadaver followed the pathway seen in the majority of cases except during the course within the epimysium of the deep surface of Flexor Digitorum Superficialis (FDS). About half way down the muscle belly, a substantial branch equal in diameter to the main trunk of the median nerve, branched off the main trunk and perforated the FDS muscle. This variant branch continued distally within the FDS muscle belly, until it reached the distal third of forearm. Then, this aberrant branch reunited with the main trunk of the median nerve as it emerged on the lateral aspect of the distal tendon of FDS (7.4cm distally from the branch point at a level 2.5cm proximal to the pisiform bone).

Conclusion

The unique variation described in this study was not found to be described in the literature. It is a reminder of the concept of patient uniqueness and the need for individualization of medical, manual, and surgical treatments offered to patients.
Variation on the Formation of Superficial Palmar Arch. A cadaveric Study
Authors: Valentin Natalia , Veras Wilson R
Published: June 2021

The anatomy of the palmar vascular arches is a complex and challenging area. Understanding the vascular patterns of the hand and their variations is very important for surgeons, radiologists and anatomists because of innovative microsurgical techniques in reconstruction of a traumatized hand, and may be useful for graft surgeries, when the arteries of the upper limb are used for coronary artery bypass. The description of a complete arch refers to the existance of an anastomosis between the superficial palmar branches of ulnar and radial arteries, or in cases in which the ulnar artery alone supplies all the fingers an the ulnar side of the thumb. An incomplete arch was defined when it only supplies the 5th, 4th, and 3rd digits. We evaluated 36 hands belonging to 19 formalin preserved cadavers to determined the pattern of the superficial palmar arch, its branches, and contributing vessels. We discard two specimen. The ulnar artery was injected with red colored latex. In addition to the frequently encountered types of superficial palmar arch, a myriad of very rare cases in which the princeps pollicis and radialis indicis arteries arising from the first and second dorsal metacarpal arteries, and from a voluminous superficial palmar branch of radial artery. In other cases the palmar metacarpal arteries gave origin to the common palmar digital arteries.
Estimating age from photographs of children's faces
Authors: DeLeon Valerie Burke , Chollet Madeleine B , Monteiro Stephen , Chung Sarita
Published: June 2021

Reuniting children separated from their families following mass disasters is a primary goal for emergency management teams. We are developing a system to facilitate this process based on automated extraction of information from photos of children collected by emergency personnel. One of our goals has been to determine the best method for estimating age of a child from such a photo, obtained under adverse field conditions where standardized collection protocols cannot be assured.

Our sample consisted of 110 children of known ages (2 mos – 16 yrs). Photos of the face were collected in frontal view. We manually collected a set of 17 landmarks using ImageJ. We developed a novel approach to establish scale in each photo based on our expectation that iris size does not vary with age. Right and left sides were averaged to reduce the effect of positioning error. Landmarks were superimposed, and we used multiple regression to test the predictive value of these facial shape variables for the estimation of age.

Our results allowed us to predict age +/− 2.5 years (95% CI). Centroid size was the most important variable in predicting age, highlighting the importance of our novel approach to establishing scale. We plan to extend these finding to landmarks that can be automatically extracted from facial photos, allowing a fast and efficient method to limit the number of photos that families must review during the reunification process.

Research was funded by HRSA EMSC Targeted Issue 5H34MC105750300.
Population Differences in Glenoid Fossa Dimensions: A 3‐D Geometric Morphometric Study
Authors: Maloney Marvelle Tekola , Pagano Anthony Santino , Marquez Samuel
Published: June 2021

The postcranial skeleton has long been studied within the context of modern human variation. Populations from cold and warm climates have been shown to exhibit shorter and longer limbs, respectively. This study tests whether the dimensions of the glenoid fossa and overall scapula also vary between populations from temperate and tropical climates. Three‐dimensional landmark coordinate data was collected from a sample of Euroamerican (EA; n=15) and Afroamerican (AA; n=15) scapulae from the division of Anthropology of the American Museum of Natural History in New York. Coordinate data from each individual was optimally scaled and superimposed as part of a Generalized Procrustes Analysis. Surface area of the glenoid fossa and the volume of humeral head accommodated within it were synthesized from interlandmark distances. It was found that the EA possessed significantly (p<0.05) taller (between the superior and inferior scapular angles) and narrower (between the acromioclavicular joint and medial boundary of the spine) scapular shape. However, there was no significant difference in the dimensions of the glenoid fossa. The results suggest that the overall proportions of the scapula share a closer relationship to limb proportion and ancestry than glenoid fossa morphology.

Grant Funding Source: SUNY Downstate Medical School
Clinical Anatomy and Variations of the Supraclavicular Veins
Authors: Schoneboom Bruce A , Rengasamy Padmanabhan
Published: June 2021

Clavicular fractures are now increasingly treated surgically. Therefore, the anatomical relationships of neurovascular structures to the clavicle assume major importance. Here we describe anatomical variants to the external jugular vein (EJV). We dissected two adult cadavers and displayed the EJV bilaterally. In both cases the EJV was found to be the inferior continuation of the retromandibular vein emerging from the inferior pole of the parotid gland. It crossed the sternocleidomastoid (SCM) muscle obliquely and pierced the deep cervical fascia to drain into the subclavian vein in the inferior medial angle of the posterior triangle. The distal end received two veins: a lateral vein about 2.00 mm in diameter crossed vertically at the anterior surface of the middle of the clavicle and drained into EJV just before the later penetrated the deep fascia; a second vein, found only on the right side arose from a network of veins at the medial edge of the sternal head of SCM, traversed laterally on the anterior surface of the clavicle and opened into the vertical vein. The inferior belly of the omohyoid and trunks of the brachial plexus were found to pass behind the EJV and posterior to the clavicle. The veins running in physical contact with the anterior surface of the clavicle might be a source of bleeding in surgical procedures involving fractures of the clavicle or procedures that use the clavicle as an anatomical landmark.
Tracking the Inferior Limit of Human Spinal Cords in Donor Bodies and Live Subjects
Authors: Golubovskaya Yevgeniya , Fisher Yuliya , Pagano Anthony S , Marquez Samuel
Published: June 2021

The human spinal cord is usually reported in medical texts as ending at the L1‐L2 vertebral levels. However, some individuals exhibit terminations at higher or lower vertebral levels. This study investigates variation in the level of caudal termination of the human spinal cord in a geriatric cadaveric sample (n=22; above 63 years of age at death) and in younger, living individuals (n=10). To determine that vertebral columns examined in donor bodies showed normal curvature, a rod was placed from external occipital protuberance (EOP) to sacral hiatus to determine the vertical line of reference (VLR) and its length. Individuals showing deviation from the VLR (i.e., scoliotic patterns) were noted. Laminectomies were performed on donor bodies to expose the vertebral canal, allowing the assignment of vertebral level to the conus medullaris. A similar protocol was followed on MR images of live subjects. All measures were collected by multiple observers to reduce bias and increase accuracy. Most (63%) of the cadavers exhibited termination at or before the superior border of L2. However, variations were present with termination levels ranging from T11‐L3 vertebrae. The live subjects expressed a similar pattern thus demonstrating that termination level of the spinal cord is independent of age. These results underscore the importance of performing spinal taps at the lowest possible vertebral levels.

Grant Funding Source: SUNY Downstate College of Medicine
Assessing Compressive Strengths and Load to Failure Limits among Vertebral Bodies: An Anatomical Study
Authors: Fisher Yuliya , Golubovskaya Yevegeniya , Hayes Westley , Saha Subrata , Marquez Samuel
Published: June 2021

The strength and compression resistance of vertebral bodies have important clinical implications with regard to susceptibility to impact trauma. This study compares compression resistance limits between the thoracic and lumbar centrums from cadaveric material to evaluate structural strength and fracture risk. The samples were radiographed to ensure healthy specimens with no evident pathologies and to document density properties (e.g., osteoporotic characteristics). Vertebral bodies were mechanically tested in compression, using the Instron 8874 (Norwood, MA) Servohydraulic Test System. Results show thoracic and lumbar strengths ranging from 0.91 N/mm2 to 1.57 N/mm2 and 0.67 N/mm2 to 1.05 N/mm2, respectively. Average maximum load to failure for the thoracic samples was 1011 N and 1620 N for lumbar samples. This study showed that while lower vertebral bodies exhibited higher load to failure values, thoracic centrums demonstrated greater maximum stress during compressive testing. Our preliminary results may shed light on the clinical understanding of compression fractures, common in the elderly, for improved outcomes.

Grant Funding Source: SUNY Downstate College of Medicine
Myology of the ocelot (Leopardus pardalis) and the significance of hindlimb myology for carnivoran phylogeny
Authors: Zack Shawn , Julik Emily , Ochoa Megan , Nair Sachin , Maredia Saaiqa , Parsa Arman , Poole Meifawn , Fisher Rebecca E.
Published: June 2021

The ocelot, Leopardus pardalis, is the most common small felid in the Neotropics. This study presents the first detailed examination of ocelot hindlimb myology, with the goals of further documenting myological diversity within Felidae and identifying characters that potentially carry a higher‐level phylogenetic signal. Hindlimbs of an adult female ocelot were dissected and documented via descriptions, photographs, and muscle maps. Data on muscular anatomy were then compared to existing descriptions of felids and added to an expanded database of hindlimb myological features in Carnivora. Results confirm previous observations that the hindlimb myology of felids is homogenous, with the exception of Acinonyx. Leopardus differs from other well‐documented felids in minor features, consistent with a very recent radiation of living felids. At the familial level, Felidae, along with Canidae and Hyaenidae are well‐defined by myological apomorphies, in contrast to most other carnivoran families. This accords well with other evidence indicating that these families are separated from their closest extant relatives by comparatively long branches, leaving considerable time for the acquisition of distinctive hindlimb myological features. At the highest level, most major clades within Carnivora (Feliformia, Caniformia, Arctoidea, Musteloidea) are supported by several potential myological apomorphies each.
The evolution of interoception: a comparison of insular and cingulate cortices in human and non‐human primates
Authors: Bethune Serene A. , Kaufman Jason A.
Published: June 2021

The insular cortex (IC) and anterior cingulate cortex (ACC) are components of a neural network involved in interoception, or awareness of the body's internal state. Previous research suggests that the cortical representation of interoceptive information in IC and ACC is unique to higher primates, including great apes and humans, and has been expanded and/or lateralized in these species. In this study, we used voxel‐based morphometry to measure the volume of IC and ACC among humans and anthropoid primates with particular interest in asymmetries and allometric deviations. T1‐weighted anatomical scans of anthropoid primate brains were acquired ex vivo using 7‐ and 9.4‐Tesla high‐field imaging systems. T1‐weighted scans of human brains were acquired in vivo using 3‐Tesla clinical imaging systems. Our data indicate that human and bonobo brains both exhibit left‐right asymmetry of the ACC in which the volume of the right ACC is significantly larger than the left. These results are consistent with known lateralization of higher cognitive functions, with the right hemisphere dominating in interoceptive tasks. The lateralization of IC and ACC could represent an important evolutionary adaptation for interoceptive capacities necessary for complex social cognition.

This research was supported by an intramural grant from Midwestern University.

Grant Funding Source: Midwestern University Intramural Grant
Temporalis muscle innervation patterns are generally conserved across subjects
Authors: Wisco Jonathan , Cantelmi David , Davies Joel , Sedlmayr Jayc , Agur Anne
Published: June 2021

The temporalis muscle is structurally complex; its muscular architecture is often misrepresented and its innervation pattern is not well characterized. In this study we digitized and mapped the 3D extramuscular and intramuscular mandibular nerve course from foramen ovale to its terminal branches in the temporalis muscle belly in four formalin embalmed cadaveric specimens. The muscle mass was dissected and digitized in layers to map its volume. The digitized nerve and muscle volume data were reconstructed into a 3D model using Maya® for visualization. Lateral 2D views were then manually registered to a common geometrical template using linear and non‐linear transformation techniques in Adobe Photoshop CS5. The extramuscular innervation consisted of three deep temporal nerves corresponding to anterior, middle and posterior portions of the muscle, masseteric and buccal nerves. Intramuscularly, the extent of muscle mass innervated by each of the nerves differed depending on the number of terminal branches. The spatial distribution of registered nerve branches was generally conserved suggesting that the degree of terminal branching was indicative of neurogenic control. We noted abundant intramuscular nerve fiber anastomosis from the anterior to posterior extent of the muscle in individuals. This work was supported by the American Association of Anatomists Visiting Scholarship.
Proteoglycan and ADAMTS expression during fibrogenic remodeling in the liver
Authors: Maurice Sean B , Crick Cody , Kim Wan‐Cheol , Law Christine , Kim Sang‐Eun , Winwood Paul J
Published: June 2021

Liver fibrosis and end stage cirrhosis are leading causes of morbidity and mortality worldwide. Changes in the structure and composition of the proteoglycan rich extracellular matrix (ECM) are known to play a role in these processes, though these changes are incompletely understood and characterized. An increased understanding of these events may one day allow early recognition and targeted therapy, leading to the reversibility of liver fibrosis as a future clinical outcome.

In other tissues, proteoglycans including versican are key components of fibrotic ECM that modulates cellular functions. The ADAMTS enzymes are now known to cleave versican and other proteoglycans in a manner previously ascribed to the MMPs. This work focuses on the role of the hepatic stellate cell in synthesizing a proteoglycan rich fibroproliferative ECM in vitro and in vivo.

We have investigated several candidate proteoglycans, including versican, along with several candidate ADAMTS enzymes, in two models of liver fibrosis. Using qPCR and confocal microscopy, we report a more thorough expression profile for these important ECM constituents than has previously been described. These findings underscore the need to better characterize the fibroproliferative liver ECM.

Grant Funding Source: Northern Medical Program
TGF‐Beta Permits Invasion of Pre‐Cancerous Keratinocytes in De‐Epidermized Dermis
Authors: Hughes Lindsay Nicole , Vaughan Melville B
Published: June 2021

Epithelial to mesenchymal transition (EMT) is an important phenotype change in the progression of carcinoma. Immortalized keratinocytes that over‐express the oncogene ras (Ker‐ct‐ras), are known from previous research to undergo EMT in vitro when put in a permissive environment. Our objective was to add Transforming Growth Factor Beta (TGF‐beta) to permit EMT in two known EMT‐inhibiting environments, De‐epidermized Dermis (DED) and liquid basement membrane skin equivalents. To test this hypothesis, Ker‐ct‐ras were plated on top of DED, then cultured in the presence or absence of TGF‐beta. The tissues were fixed at 7, 13, and 20 days for frozen and paraffin sections. Dermal invasion and protein expression changes related to EMT were evaluated, such as loss of e‐cadherin and gain of vimentin. The cells migrated from the epidermis into the dermal layer in a DED environment treated with TGF‐beta. The DED model is appropriate to study EMT and MET but is difficult to obtain and utilize, thus liquid basement membrane skin equivalents will be tested similarly. The responsiveness of Ker‐ct‐ras to environmental changes will be helpful to study gene expression changes related to cancer development.

Grant Funding Source: NCRR INBRE P20RR016478
Characterization of RTT105, a regulator of Ty1 transposition in yeast
Authors: Weir Mark Christopher , Keeney Jill
Published: June 2021

The retroelements of Saccharomyces cerevisiae include the Ty group. Ty1 is of interest because the process of transposition is very similar to how retroviruses such as HIV replicate within a cell. RTT105 is a positive regulator of Ty1 transposition identified in a genome wide screen for host genes that regulate Ty1 mobility. This gene has no characterized functional domains and shows similarity only to genes in other fungi. The aim of my research is to identify the functional domains of Rtt105. The RTT105 gene was randomly mutagenized by PCR mutagenesis, at the rate of one or two mutations per molecule, and RTT105 mutants assayed for effects on Ty1 transposition. Mutants that show Ty1 transposition frequency less than the wild type and greater than an rtt105 deletion allele are rescued and sequenced. We have also cloned RTT105 homologues from other Saccharomyces species and assayed them for transposition. These sequences and the mutant sequences were aligned. This allows us to look for conserved functional regions that may be necessary for transposition. Additionally, PSI‐Blast was performed to determine if any proteins have related sequences. A few protein sequences have been aligned with RTT105 mutant sequences. The other part of my project is to study the localization of RTT105 within a cell by confocal fluorescent microscopy. We have found Rtt105‐GFP to be localized to distinct foci in the cytoplasm. The ultimate goal is to localize Rtt105 during Ty1 transposition. This could give us some indication as to the function of the protein in regulating transposition. This research is supported by NIH grant GM074658‐01 to JBK.
Hot Start dNTPs‐ Novel chemistries for use in advanced PCR applications
Authors: Shore Sabrina , Le Tony , Ashrafi Elena Hidalgo , Timoshchuk Victor , Paul Natasha , Hogrefe Richard , Koukhareva Inna , Lebedev Alexandre
Published: June 2021

PCR is a widely used scientific tool whose specificity can be increased by the use of Hot Start technologies. Although many Hot Start technologies exist, recently developed CleanAmpTM dNTPs are a distinct approach that employs modified nucleoside triphosphates with a thermolabile protecting group at the 3′‐hydroxyl. The presence of the protecting group blocks low temperature primer extension, which can often be a significant problem in PCR. At higher temperatures, the protecting group is released to allow for incorporation by the DNA polymerase and more specific amplification of the intended target. These modified dNTPs provide comparable performance to other Hot Start technologies and can be used with a variety of thermostable DNA polymerases to turn a reaction into a Hot Start version. This thermolabile chemistry can be applied to dNTP analogs such as dUTP, which is used in UNG decontamination methods, and 7‐deaza‐dGTP, which is used to amplify difficult GC‐rich targets. In addition, further studies have led to the development of 3′‐protecting groups that deprotect more quickly than the current 3′‐modification group, allowing these modified dNTPs to be used in fast PCR. With the evolving chemistry of these Hot Start dNTPs, the areas of application benefiting from the versatility and flexibility of this technology continue to grow.
In vitro helicase loading and ORC binding on replicative and non‐replicative ACSs
Authors: Garcia‐Rivera Enrique M , Galani K , Bell Stephen P
Published: June 2021

ORC coordinates loading of the Mcm2–7 helicase complex, which leads to DNA replication. In the yeast S. cerevisiae, the origin recognition complex (ORC) recognizes a motif known as the ARS consensus sequence (ACS). Of the many ACS sites found in the yeast genome, only a small subset is actually bound by ORC. This correlates with the position of nucleosomes relative to the ACS: ChIP‐Seq analysis has shown that ORC only binds ACSs (called replicative (r) ACSs) that are within an asymmetric nucleosome free region (NFR), with precisely positioned adjacent nucleosomes and a downstream A‐rich region, most likely contributing to nucleosome exclusion. In contrast, ACSs where ORC does not bind (called non‐replicative (nr) ACSs) are located within smaller NFRs lacking the A‐rich region. We addressed the question whether nucleosome positioning dictates ORC binding and origin selection, and if sequence signatures are required in this process. Using an in vitro helicase loading assay, we showed that in the absence of nucleosomes, both rACSs and nrACSs could act as ORC binding sites and load the Mcm2–7 onto DNA. However, the rACSs load the helicase more efficiently than the nrACSs. Additionally, gel‐mobility shift assays showed that ORC has higher affinity for the rACSs. Our results indicate that sequences adjacent to the ACS play an important role in origin selection and function in the absence of nucleosomes.
Mouse models for the study of SPO11 splicing isoforms
Authors: Pratto Florencia , Bellani Marina , Camerini‐Otero Rafael Daniel
Published: June 2021

Meiotic recombination is initiated by the formation of programmed DNA double‐strand breaks (DSB) catalyzed by the SPO11 protein. This protein is widely conserved and homologs have been isolated from mouse and human. There are many alternatively spliced variants of SPO11 and two major forms (α and β) are readily detected in testes.

In males, the two major isoforms have distinct expression kinetics. Spo11 β transcripts (including all 13 exons) are found in early stages of prophase I whereas Spo11 α transcripts (exon 2 skipped) are mainly synthesized past pachytene stage. Since breaks are introduced in early prophase, the simplest model suggests that the larger form of SPO11 is the catalytic form that generates the DSB, while the smaller form has an alternative role in late prophase.

To address the role of SPO11 α we have generated an isoform specific transgenic mouse by deleting exon 2 in a BAC containing a genomic region that includes the SPO11 locus. We were able to detect overexpression of SPO11 α in a wild type mouse carrying 7 copies of the transgene and found that overepression does not affect meiotic progression.

We found that even though SPO11 α is expressed in a Spo11−/−Spo11α+/− transgenic mouse, it fails to rescue the meiotic arrest characteristic of the Spo11 KO mouse. This favors the notion of SPO11 β as the isoform responsible of introducing the breaks.

Supported by the Intramural Research Program of the NIDDK, NIH.
The PR70 subunit of PP2A regulates the levels of Cdc6 and DNA pre‐replication complex formation in U2OS cells
Authors: Sun Tingwan , Mumby Marc C
Published: June 2021

Protein phosphatase 2A (PP2A) regulates a wide variety of cellular signaling pathways including those that influence DNA replication and cell cycle. PR70 (Also known as PPP2R3B) is one of a large family of PP2A regulatory subunits previously shown to bind to the cell division control protein 6 (Cdc6), which is critical for initiation of DNA replication. The role of PR70 in the stability of Cdc6 and DNA pre‐replication complex (preRC) formation was investigated in U2OS cells. Knock‐down of PR70 by small interfering RNA (siRNA) resulted in a delay in the G1 phase of the cell cycle and a decrease in cell proliferation. Over‐expression of an siRNA resistant form of PR70 partially rescued of the cell cycle and growth phenotype of U2OS cells depleted of endogenous PR70. Loading of mini‐chromosome maintenance (MCM) proteins onto chromatin, a measure of preRC formation, was also inhibited by knockdown of PR70. The cell cycle delay and inhibition of preRC formation coincided with a reduction of the levels of Cdc6 in whole cell lysates and Cdc6 bound to chromatin. The decreased level of Cdc6 in cells depleted of PR70 appeared to be due to a combination decreased mRNA levels and accelerated degradation. In vitro assays for ubiquitination of Cdc6 by the Anaphase‐Promoting Complex‐cdh1 complex showed that PR70 reduced the ubiquitination of Cdc6. The results suggest a protective role of PR70 on ubiquitin‐mediated degradation of Cdc6 that is important for stabilizing Cdc6 and allowing formation of preRCs and cell cycle progression. This research was supported by NIH grant GM086560.
The nuclear matrix protein, Matrin 3 is required export of HIV‐1 unspliced/partially spliced RNAs
Authors: Yedavalli Venkat , Jeang Kuan‐Teh
Published: June 2021

One central aspect of gene regulation and cell functions is post‐transcriptional regulation of transcripts. Post‐transcriptional regulation involves splicing, nucleo‐cytoplasmic export of spliced RNA, stabilization‐degradation, and the translation of transcribed mRNAs. For HIV‐1, complexities exist because a single unspliced RNA must direct the synthesis of viral proteins and also serves as the genomic RNA for progeny virions. Thus unlike cellular RNAs, viral RNAs that are unspliced or partially spliced must be able to exit the nucleus and progress into the cytoplasm. To accomplish this task, HIV‐1 encodes a viral protein Rev which binds to a cis‐RNA element, RRE, which is contained on unspliced and incompletely spliced viral RNA. Rev acts to direct the export of RRE‐containing viral RNAs, and Rev has been shown to interact with cellular proteins CRM1, DDX3, PIMT and others to mediate their export. Here, we report the identification of a nuclear matrix protein, Matrin 3, as a key co‐factor which mediates Rev‐dependent expression. We discuss proposed mechanisms for how Matrin 3 acts to assist Rev function.

Research supported by Intramural funds from NIAID, and by the Intramural AIDS Targeted Antiviral Program (IATAP) from the office of the Director, NIH