Towards multiplexed immunofluorescence of 3D tissues.

Profiling molecular expression in situ allows the integration of biomolecular and cellular features, enabling an in-depth understanding of biological systems. Multiplexed immunofluorescence methods can visualize tens to hundreds of proteins from individual tissue samples, but their application is usually limited to thin tissue sections. Multiplexed immunofluorescence of thick tissues or intact organs will enable high-throughput profiling of cellular protein expression within 3D tissue architectures (e.g., blood vessels, neural projections, tumors), opening a new dimension in diverse biological research and medical applications. We will review current multiplexed immunofluorescence methods and discuss possible approaches and challenges to achieve 3D multiplexed immunofluorescence.

Bi-directional crosstalk between cells and extracellular matrix leads to network morphogenesis in multi-layered tissues.

Cell-generated mechanical forces drive many cellular and tissue-level movements and rearrangements required for the tissue or organ to develop its shape1, 2, 3, 4, 5. The prevalent view of tissue morphogenesis relies on epithelial folding resulting in compressed epithelial monolayers, overlooking the involvement of stroma in morphogenesis1, 4, 6, 7. Here, we report a giant web-like network formation of stromal cells in the epithelium-stroma interface, resulting from a multi-scale mechano-reciprocity between migrating cells and their extracellular environment. In multi-layered tissues, surface wrinkles form by a stromal cell-mediated tensional force exerted at the basement membrane. The topographical cue is transmitted to the stromal cell, directing its protrusion and migration along the wrinkles. This inductive movement of the cells conveys traction forces to its surrounding extracellular matrix, remodeling the local architectures of the stroma. In this manner, stromal cells and wrinkles communicate recursively to generate the cellular network. Our observation provides a rational mechanism for network formation in living tissues and a new understanding of the role of cellular-level tensional force in morphogenesis.

Visuomotor anomalies in achiasmatic mice expressing a transfer-defective Vax1 mutant.

In binocular animals that exhibit stereoscopic visual responses, the axons of retinal ganglion cells (RGCs) connect to brain areas bilaterally by forming a commissure called the optic chiasm (OC). Ventral anterior homeobox 1 (Vax1) contributes to the formation of the OC, acting endogenously in optic pathway cells and exogenously in growing RGC axons. Here, we generated Vax1AA/AA mice expressing the Vax1AA mutant, which is incapable of intercellular transfer. We found that RGC axons cannot take up Vax1AA protein from the Vax1AA/AA mouse optic stalk (OS) and grow slowly to arrive at the hypothalamus at a late stage. The RGC axons of Vax1AA/AA mice connect exclusively to ipsilateral brain areas after failing to access the midline, resulting in reduced visual acuity and abnormal oculomotor responses. Overall, our study provides physiological evidence for the necessity of intercellular transfer of Vax1 and the importance of the bilateral RGC axon projection in proper visuomotor responses.

Clinicopathological Significances of Positive Surgical Resection Margin after Radical Prostatectomy for Prostatic Cancers: A Meta-Analysis.

Background and Objectives: This study aims to elucidate the positive rate and the clinicopathological significance of surgical margin after radical prostatectomy (RP) through a meta-analysis. Materials and Methods: This meta-analysis finally used 59 studies, including the information about the positive surgical margin (PSM) and those clinicopathological significances after RP. The subgroup analysis for the estimated rates of PSM was evaluated based on types of surgery, grade groups, and pathological tumor (pT) stages. We compared the clinicopathological correlations between positive and negative surgical margins (NSM). Results: The estimated PSM rate was 25.3% after RP (95% confidence interval [CI] 21.9-29.0%). The PSM rates were 26.0% (95% CI 21.5-31.1%) 28.0% (95% CI 20.2-37.5%) in robot-assisted RP and nerve-sparing RP, respectively. The PSM rate was significantly higher in high-grade groups than in low-grade groups. In addition, the higher pT stage subgroup had a high PSM rate compared to the lower pT stage subgroups. Patients with PSM showed significantly high PSA levels, frequent lymphovascular invasion, lymph node metastasis, and extraprostatic extension. Biochemical recurrences (BCRs) were 28.5% (95% CI 21.4-36.9%) and 11.8% (95% CI 8.1-16.9%) in PSM and NSM subgroups, respectively. Patients with PSM showed worse BCR-free survival than those with NSM (hazard ratio 2.368, 95% CI 2.043-2.744%). Conclusions: Our results showed that PSM was significantly correlated with worse clinicopathological characteristics and biochemical recurrence-free survival. Among the results in preoperative evaluations, grade group and tumor stage are useful for the prediction of PSM.

Response to the COVID-19 Outbreak in The Emergency Department Designed for Emerging Infectious Diseases in Korea.

BACKGROUND: According to the recent coronavirus disease 2019 (COVID-19) pandemic experience, many emergency departments experience difficulties in responding to emerging infectious diseases and this has led to a public health crisis. Our emergency department (ED) is designed to respond to mass outbreaks of infection. Three major preparations were taken to respond to infectious disease; first, to improve the emergency department facilities; second, to created programs to respond to each phase of an epidemic of COVID-19; lastly, to implemented education and training to promote the safety of medical staff. We would like to share the actual responses and statistics of patients visiting emergency department during COVID-19 periods of pandemic. MATERIALS AND METHODS: This research was conducted through a retrospective chart analysis provided by a public medical center with 502 beds since the first report of a COVID-19 confirmed case on January 19, 2020 to June 15, 2020 in Seoul, the capital of Korea. Our emergency department was designed based on Korean Regional Emergency Center Facility Standards, and modified throughout each phases of COVID-19 outbreak. Patients suspected to be infectious are screened in the triage, separating them from general patients, and then receive isolation treatment in isolated wards. RESULTS: A total of 4,352 patients visited the ED. 3,202 screenings were conducted with 5 confirmed cases. Another 1,150 patients were treated with general emergent symptoms. There were no problems such as closure of the emergency department or isolation of medical staff while managing COVID-19 confirmed patients. CONCLUSION: Improving emergency department facilities, create an operational program to respond to each phase of COVID-19 outbreak and implement educational programs enabled large number of screening tests and hospitalization for COVID-19 suspected patients while maintaining general medical services. Research in emergency department designs and operational programs should increase to combine research data with better ideas to respond not only during regular periods but also during periods of pandemic.

Parental involvement in the manual reduction of pulled elbow in children.

OBJECTIVE: To investigate the effect of parental involvement in the manual reduction of pulled elbow in children. METHODS: We conducted a prospective case-control study from January to December 2018. The patients were under 6 years old with suspected radial head subluxation and were randomly assigned to two groups (an intervention group with a physician's and a parent's finger placed on the patient's radial head and a control group with the physician's finger only placed on the patient's radial head) according to the method of reduction. The results of a questionnaire given to the parent and physician were analyzed. RESULTS: A total of 150 patients were included in the study: 75 in the intervention group and 75 in the control group. There were no significant differences between the groups. The average number of attempts at manual reduction was 1.29 ± 0.73 in the intervention group and 1.31 ± 0.72 in the control group (P = 0.91) and the success rate of manual reduction was 96.0 and 94.7% (P = 0.70), respectively. The ease of conducting the procedure by the doctors did not show a significant difference between the two groups. However, the parents' understanding and satisfaction was significantly higher in the intervention group. CONCLUSION: Parental involvement did not affect the procedure of physician, but it improved the understanding and satisfaction of the parents.

Cholesterol regulates assembly of human islet amyloid polypeptide on model membranes.

Amylin, a 37-aa pancreatic hormone, is the major constituent of islet amyloid, a hallmark of type II diabetes mellitus. Recent studies have revealed a pivotal role of anionic phospholipids in membrane-catalyzed amylin fibrillogenesis and aggregation. However, cholesterol, an integral component of eukaryotic cell membranes, also could have a role. In this study, we have examined the effect of cholesterol on amylin polymerization both on planar membranes and in solution. Using time-lapse atomic force microscopy, we have studied the dynamics and macromolecular organization of amylin on anionic and neutral planar membranes that lack or include cholesterol. On cholesterol-depleted planar membranes, amylin formed highly symmetrical tetrameric and pentameric pore-like supramolecular structures composed of 25- to 35-nm intermediate-sized globular structures or oligomers. Conversely, on membranes incorporating cholesterol, amylin formed highly compact approximately 200- to 500-nm protein clusters that constituted seeds or nuclei for continuing amylin binding and aggregation. However, cholesterol inhibited amylin nucleation with a 7-fold decrease in the number of amylin particles. Consequently, cholesterol-containing membranes accumulated significantly less amyloid with some membrane areas completely free of amyloid particles. The inhibitory effect of cholesterol on amylin aggregation in solution was also demonstrated as a 16-fold decrease in the aggregation rate. Consistent with this, circular dichroism spectroscopy revealed a stable, soluble random-coil conformation for amylin in the presence of cholesterol that could explain the inhibitory effect of cholesterol on amylin polymerization in solution and on membranes. The modulatory effect of cholesterol was largely independent of membrane charge or phospholipids, suggesting a novel cholesterol-regulated amylin polymerization process.

Pituitary apoplexy presenting as isolated third cranial nerve palsy with ptosis : two case reports.

Pituitary apoplexy is a clinical syndrome caused by an acute ischemic or hemorrhagic vascular accident involving a pituitary adenoma or an adjacent pituitary gland. Pituitary apoplexy may be associated with a variety of neurological and endocrinological signs and symptoms. However, isolated third cranial nerve palsy with ptosis as the presenting sign of pituitary apoplexy is very rare. We describe two cases of pituitary apoplexy presenting as sudden-onset unilateral ptosis and diplopia. In one case, brain magnetic resonance imaging (MRI) revealed a mass in the pituitary fossa with signs of hemorrhage, upward displacement of the optic chiasm, erosion of the sellar floor and invasion of the right cavernous sinus. In the other case, MRI showed a large area of insufficient enhancement in the anterior pituitary consistent with pituitary infarction or Sheehan's syndrome. We performed neurosurgical decompression via a transsphenoidal approach. Both patients showed an uneventful recovery. Both cases of isolated third cranial nerve palsy with ptosis completely resolved during the early postoperative period. We suggest that pituitary apoplexy should be included in the differential diagnosis of patients presenting with isolated third cranial nerve palsy with ptosis and that prompt neurosurgical decompression should be considered for the preservation of third cranial nerve function.

Nano-scale imaging and dynamics of amylin-membrane interactions and its implication in type II diabetes mellitus.

Amylin, a 37-amino acid peptide hormone produced and secreted by pancreatic beta-cells, is the principal constituent of amyloid deposits in Type II Diabetes Mellitus (TTDM). Although much progress has been made in the understanding of amylin aggregation, molecular determinants that contribute to amylin aggregation in the pancreas and TTDM remain largely unknown. In order to better understand amylin aggregation and how membranes contribute to this process, visualization of amylin aggregation and deposition on membrane surface is of utmost importance. Here, we describe a new atomic force microscopy (AFM) approach to visualize amylin aggregation and to asses amylin-surface interactions. Using AFM in contact or tapping mode in fluid, amylin phase transitions on different supports were studied in real time and with high spatial nanometer-resolution. On mica, a two-stage sequential conversion of amylin from soluble monomer to small oligomers and further to mature amyloid fibrils was revealed by the AFM. This amylin conversion was accompanied by peptide conformational transition from random coil to beta-sheets assessed by CD spectroscopy. In contrast to mica, amylin formed amorphous amyloid deposits on planar lipid membranes consistent with pathological findings in diabetic subjects. Anionic lipid phosphatidylserine (PS) and membrane cholesterol had opposing effect on the kinetics and the extent of amylin aggregation. PS stimulated amylin aggregation, whereas cholesterol reversed the effect of PS. In addition, cholesterol sequestered amylin aggregates into membrane microdomains that in turn decreased amyloid deposition across the membranes. Hence, this reconstituted AFM approach offers new molecular insights to the etiology of diabetes that could be extended to investigate amylin aggregation in living islet cells at a subcellular resolution.

Regulation of the water channel aquaporin-1: isolation and reconstitution of the regulatory complex.

Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes. The molecular regulation of AQP is unknown. Here we report the isolation, identification and reconstitution of the regulatory complex of AQP-1. AQP-1 and Galphai3 have been implicated in GTP-induced gating of water in zymogen granules (ZG), the secretory vesicles in exocrine pancreas. In the present study, detergent-solubilized ZGs immunoprecipitated with monoclonal AQP-1 antibody, co-isolates AQP-1, PLA2, Galphai3, potassium channel IRK-8, and the chloride channel ClC-2. Exposure of ZGs to either the potassium channel blocker glyburide, or the PLA2 inhibitor ONO-RS-082, blocked GTP-induced ZG swelling. RBC known to possess AQP-1 at the plasma membrane, swell on exposure to the Galphai-agonist mastoparan, and respond similarly to ONO-RS-082 and glyburide, as ZGs. Liposomes reconstituted with the AQP-1 immunoisolated complex from solubilized ZG, also swell in response to GTP. Glyburide or ONO-RS-082 abolished the GTP effect. Immunoisolate-reconstituted planar lipid bilayers demonstrate conductance, which is sensitive to glyburide and an AQP-1 specific antibody. Our results demonstrate a Galphai3-PLA2 mediated pathway and potassium channel involvement in AQP-1 regulation.