Excess Stain Research Articles

(024) FIRST TIME ASSESSMENT OF HYMENAL TISSUE IMMUNOHISTOCHEMICAL STAINING FROM PATIENTS WITH NEUROPROLIFERATIVE VESTIBULODYNIA

Abstract Introduction Embryologically, the hymen forms at the junction of the endodermal urogenital sinus and the mesodermal primitive vagina. During fetal development the hymen becomes surrounded by the endodermal vestibule distally and the endodermal vagina proximally (Fig. 1) and therefore, is likely to also be of endodermal origin. Neuroproliferative vestibulodynia (NPV) is a provoked vestibular pain disorder associated with excess immunohistochemical (IHC) staining with CD117, a mast cell marker, throughout the entire 1:00-11:00 o’clock as well as 12:00 o’clock vestibular regions. Although the hymen is surrounded by endodermal tissue, to the best of our knowledge, it has not been established whether hymenal tissue of patients with NPV also would have excess IHC staining consistent with mast cells. This is clinically relevant, as some patients have persistent post-operative pain in the region of the unexcised hymen when vestibulectomy is not complete. Objective We wished to assess, for the first time, CD 117 immunostaining in the hymenal tissue of NPV patients and compare the IHC data with that of excised vestibular tissue. Methods Medical records from the patient cohort who underwent complete vestibulectomy between April 1, 2024 and May 31, 2024 were reviewed. For this patient cohort, three separate specimens were sent to pathology for analysis: i) 1:00-11:00 vestibule, ii) 12:00 vestibule and iii) hymenal tissue that had been sharply separated from the 1:00-11:00 excised vestibular specimen (Fig. 2). Slides of IHC-stained vestibular tissue specimens from the 1:00-11:00 and 12:00 vestibule as well as hymen were examined, and images captured using a 3.5 megapixel digital camera attached to a light microscope at 100× magnification. A minimum of 2 photomicrographs were taken for each stained slide, identifying representative regions of the epithelial basement membrane and adjacent subepithelium. A total of 48 photomicrographs of slides stained for CD117 were assessed. Each photomicrograph was assigned a unique identification number. Manual counting of CD117-immunopositive stained cells was performed on these photomicrographs for comparison to Bornstein’s data that >8 mast cells per high power field confirmed the diagnosis of NPV. Results A total of 8 NPV patients (mean age 28 years, range 21–42) were included in the chart review. All had symptoms consistent with NPV, and all had other conditions associated with aberrant mast cell activity. Both 1:00– 11:00 and 12:00 vestibular tissue specimens had >8 mast cells per high power field. The hymenal tissue in all 8 patients also contained >8 mast cells per field at the same magnification (Fig. 3). Median manual mast cell count values were similar in 1:00-11:00 o’clock and 12:00 vestibular regions and the hymen: 45 (range 21-67), 39 (range 19-63) and 43 (range 18-68) mast cells per 100× field, respectively. Conclusions NPV is a field disease of the endoderm, which based on these data, includes all hymenal tissue. To maximize success from complete vestibulectomy for NPV, excision of all hymenal tissue should be considered. Disclosure No.

(027) PREVALENCE OF CONDITIONS ASSOCIATED WITH ABERRANT MAST CELL ACTIVITY IN PATIENTS WITH NEUROPROLIFERATIVE VESTIBULODYNIA

Abstract Introduction Neuroproliferative vestibulodynia (NPV) is a provoked vestibular pain condition associated with excess immunohistochemical staining for CD117 and PGP9.5, consistent with mast cells and nerves respectively. It is distinguished from control vestibular tissue by having >8 mast cells/high power field. NPV is thus a condition associated with aberrant mast cell activity. Objective In patients with histopathologically confirmed NPV, we assessed prevalence of other conditions involving aberrant mast cell activity. Methods Medical records from a single facility of patients who underwent a vestibulectomy between June 1, 2019 and May 31, 2024 were reviewed. All patients had histopathologically confirmed NPV. Evidence of other conditions of aberrant mast cell activity was collected based on the medical history form completed by each new patient as well as the history recorded during the initial patient interview, including the following: asthma, chronic sinusitis, endometriosis, food allergy/intolerance, interstitial cystitis (IC), irritable bowel syndrome (IBS), migraine, and postural orthostatic tachycardia syndrome (POTS). That list has been expanded in the last 9 months to include other conditions of aberrant mast cell activity including: seasonal allergies, joint hypermobility, gastroesophageal reflux disease (GERD) and temporomandibular joint (TMJ) dysfunction. Results A total of 113 NPV patients (mean age 29, range 19-64 years) were included in this chart review. No other aberrant mast cell activity was reported by 38 (34%), however 75 (66%) had at least one concomitant condition. Of the individuals with other mast cell conditions, the most common were: migraine (n = 29; 26%), IC (n = 25; 22%), endometriosis (n = 20; 18%), food allergy/intolerance (n = 18; 16%), IBS (n = 14; 12%), asthma (n = 9; 8%), chronic sinusitis (n = 5; 6%) and POTS (n = 3; 3%). After assessing for a broader spectrum of conditions of mast cell aberrant activity, additional disorders reported in the last 25 patients with NPV include seasonal allergies (n = 5; 20%), hypermobility (n = 5; 20%); GERD (n = 4; 16%), TMJ (n = 4; 16%). Among this cohort of 113 patients, a total of 40 (35%), 17 (15%), 6 (5%), 5 (4%) and 4 (4%) had one, two, three, four and five or more other conditions involving aberrant mast cell activity, respectively. There was no correlation between the number of concomitant conditions with aberrant mast cell activity reported and the density of CD117-immunopositive staining in the overall NPV cohort. Conclusions Patients with histopathologically confirmed NPV have a high prevalence of concomitant conditions associated with aberrant mast cell activity, possibly due to a genetic predisposition. Based on this experience, patients with vulvas are now queried using a more expansive list of conditions of aberrant mast cell activity, as NPV is likely part of a broader systemic collection of these conditions. This finding may be relevant for future diagnostic and treatment options for patients with NPV. Disclosure No.

(130) PREVALENCE OF CONDITIONS INVOLVING ABERRANT MAST CELL ACTIVITY IN PATIENTS WITH NEUROPROLIFERATIVE VESTIBULODYNIA

Abstract Introduction Neuroproliferative vestibulodynia (NPV) is a provoked vestibular pain shown to be associated with excess immunohistochemical staining for CD117 and PGP9.5, consistent with mast cells and nerves, respectively. NPV is distinguished from control tissue by having >8 mast cells/high power field. Objective In patients with a histopathologically confirmed diagnosis of NPV, we wished to assess the prevalence of concomitant conditions involving other aberrant mast cell activity. Methods Medical records from people with vulvas who underwent vestibulectomy between June 1, 2019 and October 31, 2023 at a single facility were retrospectively reviewed. All had histopathologically confirmed NPV, having observed > 8 positive CD117 stained cells/high power field in the sub-epithelial stroma. As part of their history, each patient provided a list of medical conditions which included disorders where aberrant mast cell activity has been reported. The following eight conditions were included in medical history forms: asthma, chronic sinusitis, endometriosis, food allergy/intolerance, interstitial cystitis, irritable bowel syndrome, migraine, and postural orthostatic tachycardia syndrome. Results A total of 88 NPV patients were included in this chart review. A total of 34 (39%) reported no other aberrant mast cell activity, however 54 (61%) had at least one concomitant condition. The most common conditions were migraine (n=21; 24%), interstitial cystitis (n=17; 19%) and endometriosis (n=15; 17%), followed by food allergy/intolerance (n=12; 14%), irritable bowel syndrome (n=9; 10%), asthma (n=9; 10%), chronic sinusitis (n=5; 6%) and postural orthostatic tachycardia syndrome (n=2; 2%). Among these 88 patients, a total of 33 (38%), 13 (15%), 3 (6%), 1 (1%) and 2 (2%) had one, two, three, four and five other conditions involving aberrant mast cell activity, respectively. There was no correlation between the number of concomitant conditions with aberrant mast cell activity reported and the density of CD117-immunopositive staining in the overall NPV cohort, whether diagnosed with lifelong or acquired NPV. Conclusions Patients with histopathologically confirmed NPV have a high prevalence of concomitant conditions associated with aberrant mast cell activity, possibly due to a genetic predisposition. While more studies are needed, NPV is likely part of a broader systemic collection of conditions associated with aberrant mast cell activity. This finding is relevant in terms of future diagnostic and treatment options for patients with NPV. Disclosure No.

(267) The Prevalence of Conditions Involving Aberrant Mast Cell Activity in Patients with Neuroproliferative Vestibulodynia

Abstract Introduction Neuroproliferative vestibulodynia (NPV) is a provoked vestibular pain, shown to be associated with excess immunohistochemical staining for CD117 and PGP9.5, consistent with mast cells and nerves, respectively. It is hypothesized that NPV occurs in genetically predisposed patients in whom mast cells accumulate in the vestibular sub-epithelial stroma where they release various cytokines/growth factors that stimulate the growth of nerves, causing severe allodynia and hyperalgesia throughout the entire vestibule. Objective To assess, in our patient cohort with histopathologically confirmed diagnosis of NPV, the prevalence of concomitant conditions involving aberrant mast cell activity. Methods Medical records from people with vulvas who underwent vestibulectomy between June 1, 2019 and December 31, 2022 were retrospectively reviewed. This research protocol was IRB-exempt. Histories from these patients, all of whom had histopathologically confirmed NPV after observing > 8 immunopositive CD117 stained cells per high-power field in the sub-epithelial stroma, were reviewed. The prevalence was noted for the presence of concomitant conditions where aberrant mast cell activity has been reported to play a critical role in symptomatology. The focus was on the following eight conditions: asthma, chronic sinusitis, endometriosis, food allergy/intolerance, interstitial cystitis, irritable bowel syndrome, migraine, and postural orthostatic tachycardia syndrome. Results A total of 65 NPV patients were included in this chart review. While 24 (37%) reported none, 41 (63%) had at least one other concomitant condition involving aberrant mast cell activity. A total of 25 (38%), 10 (15%), 4 (6%) and 2 (3%) had one, two, three and five other conditions, respectively, involving aberrant mast cell activity. The most common such condition was migraine (n=14; 22%), followed by endometriosis (n=12; 19%), food allergy/intolerance (n=11; 17%), interstitial cystitis (n=10; 15%), irritable bowel syndrome (n=8; 12%), asthma (n=7; 11%), chronic sinusitis (n=5; 8%) and postural orthostatic tachycardia syndrome (n=2; 3%). There was no correlation between the number of concomitant conditions with aberrant mast cell activity reported and the density of CD117-immunopositive staining in the overall cohort of patients with NPV or within the subgroups of those with either lifelong or acquired NPV. Conclusions Patients with histopathologically confirmed NPV have a high prevalence of concomitant conditions associated with aberrant mast cell activity. While more studies are needed, NPV is likely part of a broader systemic collection of conditions associated with aberrant mast cell activity. This finding is relevant in terms of future diagnostic and treatment options for patients with NPV. Disclosure No.

Taxonomic significance of leaf architecture in the genus Basella Linn. in Nigeria

This study seeks to use the leaf architecture as a taxonomic tool for members of the genus Basella Linn. in Nigeria, namely, B. rubra Linn., B. alba Linn., B. cordifolia Lamk. and a form, B. alba round because the taxonomy of the genus Basella Linn. is not clear and there is no information on the leaf architecture of the genus. For studies of the venation pattern of the leaves, sizeable portions were taken from the standard median portion of mature leaves. Cleared leaves were stored in 50% alcohol for anatomical studies. Leaves were stained in Safranin O for 3 minutes and counterstained in Alcian blue and then were rinsed in water to remove excess stain. Stained cleared leaves were treated in serial grades of alcohol for differentiation and dehydration. Stained materials were mounted in dilute glycerol for microscopic examination. Photomicrographs were taken with the aid of 3013 ACCU-SCOPE Trinocular Microscope with Digital Camera. The areolar area was calculated from the length and breadth of the areoles measured using a micrometer inserted into the microscope eyepiece. Basella species have common generic features. Areoles are closed in B. rubra but others have veinlet endings. The area of areole is diagnostic because in B. rubra the area is 305,370±1808.91 μm2; B. alba is 501,796.75±3217.38 μm2, B. cordifolia’s area of areole is 396,394.75±2670.67 μm2 while that of B. alba round is 540,058.75±3702.28 μm2. The arrangement of druses in the areoles is also diagnostic.

In vitro reduction of Escherichia coli biofilm mass by platelet-rich plasma lysate (PRP-L)

Placentitis-induced, pre-term fetal loss has caused hardships and financial losses to the equine breeding industry for decades. While traditional multi-modal treatments including a combination of gentamicin, penicillin, altrenogest, firocoxib, and pentoxifylline can sometimes prolong gestation and neonatal survival, treatment failures still occur and antibiotic therapy can be quite lengthy. Understanding potential biofilms in these reproductive infections uncovers a pitfall to the success of traditional treatment options: antimicrobials are ineffective at penetrating and killing biofilms in vitro and in vivo. Platelet-rich plasma lysate (PRP-L) is increasingly accepted as an adjunctive treatment option for biofilm associated infections across species. The aim of this study was to evaluate the reduction in biofilm formation of 10 isolates of Escherichia coli collected from the equine uterus. Isolates were stored in glycerol at -80°C; each isolate including a positive (ATCC 25922) and negative (ATCC BAA 2469) strain, was cultured for 18-24 h at 33-37°Cin ambient air until bacterial colonies were visible. A suspension of each isolate, adjusted to a 0.5 McFarland value, was used to inoculate a tissue culture treated, round-bottom 96-well microtiter plate incubated under microaerophilic orbital shaking conditions for 18-24 h at 33-37°C. Following incubation, with or without PRP-L, the supernatant was removed, wells were stained with 0.03% crystal violet for 15 minutes at room temperature with orbital shaking, rinsed to remove excess stain, solubilized with ethanol, and absorbance optical density (OD570) read with a plate reader. The range in reduction of biofilm mass with PRP-L treatment was 48 – 84%, with an average of 60%. In conclusion, the results suggest that comparison of a traditional multi-modal therapy for placentitis with a multi-modal therapy augmented with PRP-L has the potential to finally improve placentitis outcomes and reduce the length of antibiotic treatment, but further in vivo evaluation of this approach is required.

Comparative Anatomical Studies of Four Accessions of Bambusa vulgaris Schrad. ex J.C. Wendl. Collected in Ile-Ife, Osun State, Nigeria

A comparative anatomical study was carried out on four accessions of Bambusa vulgaris to identify characters of taxonomic purposes to better understand the relationship between the four accessions and also to fill the knowledge gap in the understanding of their taxonomy. Transverse sections, tangential longitudinal sections and radial longitudinal sections of mature leaf, stem and roots were cut at 8-15 µm on the Reichert sliding microtome. Some sections were done by hand for preliminary studies. Each section was stored in 50% alcohol for anatomical studies. The sections were stained in Safranin-O for three minutes, rinsed in water to remove excess stain and counterstained in Alcian blue for three minutes. Later, the sections were rinsed in water to remove excess stain and then treated with serial grades of alcohol and mounted in dilute glycerol for study. Photomicrographs were taken with the aid of 3013 ACCU-SCOPE Trinocular Microscope with Digital Camera. The qualitative investigations of the four accessions revealed that they were similar. However, accession 1 forms a sister clade with accession 3 and accession 4. This research concluded that quantitative stem and root anatomical characters are important in delineating species accessions and should be employed in separating closely related species as well as different accessions of the same species.

In vitro anti-oxidant and anti-adhesin assays of n-hexane and methanolic extracts of Caryota no seeds

Abstract To determine the anti-oxidant and anti-adhesin activities of the n-hexane and methanolic extracts of Caryota no (CN) seeds. For anti-adhesin properties, the wells of a sterile flat-bottomed tissue culture plate were filled with 200µL of each extract in several concentrations, incubated and washed twice. 200µL of bacterial suspension was added, incubated and washed thrice with PBS. Adherent bacteria were fixed with methanol, plates emptied, left to dry and stained with crystal violet. Excess stain was rinsed under running water, air dried, bound dye resolubilized with glacial acetic acid, and the absorbance measured using a microplate reader to calculate the percentage inhibition. For antioxidant properties, three different concentrations of the DPPH solution were prepared. 2 mg of each extract was weighed out and dissolved in 10 mL of their corresponding solvent. 1 mL of DPPH solution was added to 2 ml solution of the extracts and ascorbic acid. The reaction mixture was vortexed and left in the dark for 30 minutes then absorbance was measured against blank solution to calculate the percentage scavenging radical inhibition. Statistical difference was presumed at P < 0.05. The extracts showed significant (P < 0.05) adhesin inhibition activities, n-hexane (64%) and methanolic (46%) at 200µg/mL concentrations. The fractions of the extracts also showed various remarkable results. The anti-adhesin activity increased with increasing extract concentration in both extracts. Antioxidant assays revealed that at 200 mg/mL, both extracts were significantly (P < 0.05) lower than the control. Both the n-hexane and methanolic extracts showed remarkable adhesin inhibition activities at concentration of 200 µg/mL and antioxidant effects, with the methanolic extract demonstrating comparable levels as the standard, ascorbic acid. This implies that both extracts may serve as veritable natural sources of anti-adhesins, antioxidants and thus adjunct anti-infective agents or immune enhancers. Keywords DPPH, Caryota no, Adhesin, Reactive oxygen species, anti-oxidant

Topical Henna Application Causing Acute Kidney Injury and Acute Hemolytic Anemia.

Body art by henna staining is a practice that is widely prevalent in the Middle East and Africa, and has been known to be in vogue for hundreds of years. The practice is also significant as a ceremonial custom for weddings and social gatherings. However, due to its natural components (Lawsone) and additives, including para-phenylene-diamine (PPD), henna has also been associated with a number of health hazards, including acute hemolysis and acute kidney injury (AKI). We report in this case, a female patient who presented with AKI and acute hemolytic anemia following excessive pre-wedding henna staining of her arms and legs.

Is Conventional Management of Hyperpigmentation Still the Best Choice? -- A Case Report

Background: A normal gingiva is generally “salmon” pink in color. Varying degrees of brownish pigmentation in gingiva may be seen in Blacks (seldom also in Caucasians). Those who exhibit gingival hyperpigmentation face aesthetic problems. Objective: To describe the treatment of gingival hyperpigmentation in a 29-yearold male attending the Department of Periodontics Clinic, Universitas Padjadjaran, exhibiting excessive staining on the gums of the right to left canine region of the upper and lower jaw. Case Report: This gingival hyperpigmentation case was managed through a conventional method of using scalpel. Results: Gingiva that had excessive staining in this case was eliminated and exhibited good aesthetic. Conclusion: The conventional management of gingival hyperpigmentation is still the best choice to solve an aesthetic problem with a simpler and safer technique, and at more affordable costs. Keywords: gingival hyperpigmentation, gum excessive staining, conventional gingival surgery

Comparative Image Quality Analysis of Spatial Filters for Pre-processing of CT Abdominal Images

Aim: To determine the efficient noise reduction filter for abdominal CT images. Background: Image enrichment is the first and foremost step that has to be done in all image processing applications. It is used to enhance the quality of digital images. Digital images are liable to addition of noise from various sources such as error in instrument calibration, excess staining of images, etc., Image de-noising is an enhancement technique used to remove / reduce noise present in an image. Reducing the noise of images and preserving its edges are always critical and challenging in image processing. Materials and Method: In this paper, four different spatial filters namely Mean, Median, Gaussian and Wiener were used on 100 CT abdominal images and their performances were compared against the following four parameters: Peak signal to noise ratio (PSNR), Mean Square Error (MSE), Normalised correlation coefficient (NCC) and Normalised Absolute Error (NAE) to determine the best denoising filter for the abdominal CT images. Result: Based on the experimental parameters, the median filter had the maximum efficiency in managing salt and pepper noise than the other three filters. Both Median and Wiener filters showed efficiency in removing the Gaussian noise. Whereas, the Wiener filter demonstrated higher efficiency in reducing both Poisson and Speckle noise. Conclusion: Based on the results of this study, we can conclude that the median filter can be used to reduce Salt and Pepper noises. Median and Wiener filters are significantly better for Gaussian Noise and the Wiener filter can be used to reduce both Poisson &amp; Speckle noise in abdominal CT images.

To evaluate if ultrasound gel or water-based media affects ER/PR staining patterns on breast core biopsy specimens

Abstract Introduction/Objective IHC staining for biomarkers ER, PR and HER-2 (CerB2) forms an essential component of breast cancer management and prognostication. Staining for these biomarkers is dependent on optimal fixation of the specimen in formalin. Scoring systems employed for ER and PR grade the intensity of the nuclear staining along with the percentage of the cells stained. This study aims to determine if ultrasound gel used during core biopsy as opposed to water affects immunohistochemical staining of biomarkers ER and PR in breast cancer specimens, following an observation that ultrasound gel assisted core biopsies may affect ER/PR nuclear staining scores through excess cytoplasmic staining. Methods/Case Report We included a total of 50 cases of invasive breast carcinoma diagnosed on core biopsy from KK Women’s and Children’s Hospital from February 2019 to July 2020. These included 25 cases with gel and 25 cases with water applied to the breast prior to the procedure. All biopsies were optimally fixed in formalin as per recommended latest ASCO/CAP guidelines. The control group included 25 resection cases of invasive breast carcinoma which had neither gel nor water and had been optimally fixed in formalin. Cytoplasmic staining for ER and PR was graded as being present or absent and was compared against the excision specimens (control group) to determine if using the gel was significant in producing cytoplasmic staining. The data was analyzed on SPSS using multiple logistic regression. Results (if a Case Study enter NA) Using the gel appeared to be statistically significant in producing cytoplasmic staining for ER (p=0.042) as opposed to using water (p=0.274). For PR staining, both gel-based (p=0.250) and water-based media (p=0.286) were not statistically significant in causing cytoplasmic staining. Conclusion The preliminary findings of this small retrospective case-control study gives an insight that gel-based media may have a role in causing cytoplasmic staining for oestrogen receptor in breast cancer biopsy specimens which may interfere with accurate scoring and impact results of external quality assurance schemes to which laboratories subscribe to. However the study is limited by the small sample size and exclusion of any other pre- analytical factors like needle bore or number of cores taken which may have had an impact on the staining.

Laparoscopic Management of Type 2 Cesarean Scar Pregnancies - Planning and Procedural Execution

<h3>Study Objective</h3> With increasing caesarean deliveries, Caesarean scar pregnancies CSP are also on the rise. We report our experience from successful laparoscopic management of type 2 exogenic CSPs which invades deep, projects into the vesicouterine fold and has the dangerous potential for rupture and massive hemorrhage. <h3>Design</h3> NA. <h3>Setting</h3> tertiary referral center. <h3>Patients or Participants</h3> four women confirmed to have type 2 cesarean scar pregnancy. <h3>Interventions</h3> MRI was done for preoperative planning to clearly define the sac, note dehiscence of scar and adherence of uterine corpus to anterior abdominal wall. This spatial resolution gave a good mental picture. At Laparoscopy, first adhesions are released and round ligaments divided temporarily, giving better access to lower uterine segment for surgery and suturing. Prior to making an incision on the uterus, the complete scar with sac inside is exposed till the anterior cervix. Careful controlled dissection avoids excessive bleeding and staining, which could result in loss of anatomic orientation and even panic. Bilateral uterine artery occlusion and Local injection of vasopressin are interventions used for decreasing bleeding from otherwise highly vascular gravid uterus. After complete resection and removal of the products, Full thickness, equidistant repair of LUS defect is aided by barbed suture. A Hegar's dilator placed in situ during the suturing phase prevents accidental approximation of uterine walls. Here we humbly acknowledge that we have managed laparoscopically one of the most advanced CSP reported so far at 16 weeks gestational age. <h3>Measurements and Main Results</h3> all four women had procedures accomplished laparoscopically, with complete resection of the type 2 CSPs and satisfactory reconstruction of defect, also received the benefits of the minimally invasive surgery such as less pain, less scar and rapid return to work. <h3>Conclusion</h3> Laparoscopy here is proven safe and effective, in view of excellent visualization, complete removal of pathology, good reconstruction of the defect, and should be the choice where facilities exist.

Effectiveness and Safety of Posterior Vaginal Repair with Single-Incision, Ultralightweight, Monofilament Propylene Mesh: First Evidence from a Case Series with Short-Term Results.

Objective The use of transvaginal mesh is controversial, and over time, multiple surgical methods for the treatment of posterior vaginal prolapse (PVP) have been proposed including different surgical approaches and techniques. To date, no clear conclusion has been reached about the use of mesh for reinforcing transvaginal posterior repair. The aim of this study was to evaluate the feasibility, safety, and effectiveness of a novel, ultralightweight mesh for the treatment of PVP. Methods We performed a single-center, prospective observational study on consecutive patients referred for primary or recurrent, symptomatic stage II PVP (according to the international Pelvic Organ Prolapse Quantification System) from April 2017 to September 2018. In all patients, transvaginal posterior repair was augmented with a single-incision, isoelastic polypropylene mesh. Data about the postoperative outcomes were collected until December 2019. Results A total number of 15 patients were included. The median follow-up after surgery was 18 months (IQR = 14). Surgery was completed in all cases without complications. Regarding the anatomical outcomes (as measured according to POP-q classification), a significant improvement was observed in terms of Bp, D, and C (p < 0.05). The functional outcomes were significantly ameliorated after surgery, with a reduction of bulge symptom, stypsis, incomplete evacuation, and excessive staining (p < 0.05). The quality of life was significantly improved in the majority of patients (p < 0.05). Median patients' satisfaction rate was 100% (IQR = 22.5%). Neither early nor late postoperative complications occurred. Conclusions Single-incision, ultralightweight polypropylene meshes were safe and highly effective in the treatment of PVP. As our study has some limitations, further large, controlled studies are needed.

PD-L1 immunohistochemistry in non-small-cell lung cancer: unraveling differences in staining concordance and interpretation

Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) is accepted as a predictive biomarker for the selection of immune checkpoint inhibitors. We evaluated the staining quality and estimation of the tumor proportion score (TPS) in non-small-cell lung cancer during two external quality assessment (EQA) schemes by the European Society of Pathology. Participants received two tissue micro-arrays with three (2017) and four (2018) cases for PD-L1 IHC and a positive tonsil control, for staining by their routine protocol. After the participants returned stained slides to the EQA coordination center, three pathologists assessed each slide and awarded an expert staining score from 1 to 5 points based on the staining concordance. Expert scores significantly (p < 0.01) improved between EQA schemes from 3.8 (n = 67) to 4.3 (n = 74) on 5 points. Participants used 32 different protocols: the majority applied the 22C3 (56.7%) (Dako), SP263 (19.1%) (Ventana), and E1L3N (Cell Signaling) (7.1%) clones. Staining artifacts consisted mainly of very weak or weak antigen demonstration (63.0%) or excessive background staining (19.8%). Participants using CE-IVD kits reached a higher score compared with those using laboratory-developed tests (LDTs) (p < 0.05), mainly attributed to a better concordance of SP263. The TPS was under- and over-estimated in 20/423 (4.7%) and 24/423 (5.7%) cases, respectively, correlating to a lower expert score. Additional research is needed on the concordance of less common protocols, and on reasons for lower LDT concordance. Laboratories should carefully validate all test methods and regularly verify their performance. EQA participation should focus on both staining concordance and interpretation of PD-L1 IHC.

A simple, economical and environmental-friendly method for staining protein gels using an extract from walnut-husk

We wish to present a simple, rapid, cost-effective and environmentally safe method for staining proteins in polyacrylamide gels, using aqueous-based natural extracts from fresh green walnut (Juglans regia) hulls/husks. The technique takes not more than 10 min for staining and is comparable in sensitivity to the most commonly used Coomassie R-250 staining method when applied to different concentrations of Bovine Serum Albumin (BSA) and various amounts of E. coli extracts. The protein (BSA) band (~0.5 μg) and E. coli extract comprising ~25 μg total protein can be visualized on polyacrylamide gels. Compared to both Coomassie and Ponceau S staining, the current method displayed more intense bands when proteins are transferred to polyvinylidene fluoride (PVDF) membrane. Although the walnut-dye (WD) method does not require a time-consuming destaining step, excess background stain can simply be removed by washing in water. Extract from old dried black husks and extract from fresh green husks kept for a year was also effective. Using LC-MS, Myricetin and/or Kaempferol were found to be active compounds responsible for staining proteins. Compared to traditional Coomassie method, the inclusion of expensive and toxic solvents (methanol and acetic acid) is completely avoided resulting in positive health, environmental and economic benefits. In view of all these advantages, the WD method has immense potential to replace currently used protein staining techniques.

Core Binding Factors are essential for ovulation, luteinization, and female fertility in mice

Core Binding Factors (CBFs) are a small group of heterodimeric transcription factor complexes composed of DNA binding proteins, RUNXs, and a non-DNA binding protein, CBFB. The LH surge increases the expression of Runx1 and Runx2 in ovulatory follicles, while Cbfb is constitutively expressed. To investigate the physiological significance of CBFs, we generated a conditional mutant mouse model in which granulosa cell expression of Runx2 and Cbfb was deleted by the Esr2Cre. Female Cbfbflox/flox;Esr2cre/+;Runx2flox/flox mice were infertile; follicles developed to the preovulatory follicle stage but failed to ovulate. RNA-seq analysis of mutant mouse ovaries collected at 11 h post-hCG unveiled numerous CBFs-downstream genes that are associated with inflammation, matrix remodeling, wnt signaling, and steroid metabolism. Mutant mice also failed to develop corpora lutea, as evident by the lack of luteal marker gene expression, marked reduction of vascularization, and excessive apoptotic staining in unruptured poorly luteinized follicles, consistent with dramatic reduction of progesterone by 24 h after hCG administration. The present study provides in vivo evidence that CBFs act as essential transcriptional regulators of both ovulation and luteinization by regulating the expression of key genes that are involved in inflammation, matrix remodeling, cell differentiation, vascularization, and steroid metabolisms in mice.

Anti-mutagenic Action of Ruta chalepensis against Rat Sperm Cell Abnormalities-induced by Potassium Bromate

Aims: The aim of current study was to investigate the genotoxic damage of potassium bromate on sperm cell morphology and the potential defense role of Ruta chalepensis (Rue) against the potassium bromate effects.&#x0D; Study Design: Fifty rats (Strain: Rattus norvegicus) were randomly divided into five groups (10 rats/group) as follows: (Control group) received distilled water daily for 4 weeks, (KBrO3 group) treated with KBrO3 (100 mg/kg/b.w) for 4 weeks, (Rue group) received Rue orally in a daily dose (0.5 g/animal) for 4 weeks, (treatment group) were first provided with oral doses of KBrO3 daily for 2 weeks and then treated orally with KBrO3 in association with Rue for an additional 2 weeks Finally (protective group) was administered with Rue orally for 2 weeks and then administered oral doses of Rue in association with KBrO3 for an additional 2 weeks.&#x0D; Place and Duration of Study: Department of Zoology and Department of Botany, University of Omar Almukhtar, between July 2018 and September 2019.&#x0D; Methodology: Sperm suspensions, filtered and smears were made according to the standard protocol for sperm morphology assay. The smears were fixed, allow to dry and stained with hematoxylin and then washed under slow running water until the excess stain is removed and were putted in eosin dye.&#x0D; Results: The results were showed that potassium bromate has the ability to cause mutations in the morphology of sperm cells. The highest mutation rates of head, tail and neck reached 16.60, 5.200 and 3.200 respectively in animals. Several types of sperm abnormalities such as hock free, banana type, amorphous, hummer shape after the administration of potassium bromate comparing with the negative control animals that treated only with potassium bromate. On the other hand, the oil extract of Ruta chalepensis significantly inhibited the mutations by declining the abnormality rates of head, tail and neck to 2.600, 1.600 and 0.400 in protective group. Thus, potassium bromate has to be considered as a conceivable line regarding human health.&#x0D; Conclusion: Potassium bromate clearly caused a wide range of sperm cell abnormalities and Rue oil extracts have a good anti-mutagenic effects against potassium bromate by decreasing the sperm cell abnormalities.

Course of loss of photoreceptor function and progressive Müller cell gliosis in rhodopsin P347L transgenic rabbits

Long living animal models of retinitis pigmentosa (RP) can provide important information on the retinal changes that occur at the late stages of photoreceptor degeneration. The rhodopsin Pro347Leu transgenic rabbit (P347L Tg) is a model of RP, and it has been used to analyze the functional and morphological changes in the retina following the degeneration of the photoreceptors. They have also been used to test newly-developed therapies to treat eyes with photoreceptor degeneration. However, assessments of the retinal changes in P347L Tg rabbits older than 1-year have not been reported even though the data are important for research on developing new therapies to restore vision at the end stages of RP. The purpose of this study was to determine the time course of the loss of photoreceptor function and the changes in the morphology of the retina of P347L Tg rabbits. The experiments were performed on 26 older P347L Tg rabbits. The results showed that the amplitudes of the ERGs of the P347L Tg rabbits gradually decreased and reached <10 μV between 30- and 54-months-of-age. Histological analysis at these later stages showed a loss of the photoreceptor layer, and OCT analysis showed absence of the layering of the retina. However, the thickness between the inner limiting membrane and the outer plexiform layer was about 1.7 times thicker than the corresponding thickness of WT rabbits in the OCT images. This thickening was caused by a marked gliosis of the entire retina which was confirmed by light and transmission electron microscopy. In addition, immunohistochemical analysis showed there was excessive staining of the glial fibrillary acid protein in the older P347L Tg rabbits although the rod ON bipolar cells and horizontal cells were still present in the inner nuclear layer. Our results indicate that the P347L Tg rabbit progressed to complete photoreceptor loss within 30- and 54-months-of-age and severe gliosis altered the morphology of the retina.

Large number tracking of depth and mechanics dependent calcium signaling in chondrocytes of articular cartilage

Purpose: Moderate loading of cartilage stimulates chondrocytes to synthetize matrix proteins, but excessive loading can lead to apoptosis. It is well known that macroscale strains affect cell health, but the mechanism behind how tissue level strain causes cellular and subcellular biological response is poorly understood. Previous work done by our group showed impact-induced tissue strain resulting in mitochondrial depolarization in chondrocytes, with correlation between strain and chondrocyte death, demonstrating the importance of microscale mechanics driving chondrocyte fate after impact. The correlation between strain and mitochondrial dysfunction indicates the mitochondria’s role in mechanosensing. However, the mechanotransduction and cell signaling pathway driving this process has not been fully characterized. In traumatic brain injury, Ca2+ signaling is known to produce mitochondrial swelling, while in other tissues Ca2+ is buffered by mitochondria. However, if too much Ca2+ is taken up, the mitochondrial permeability transition pore will open, leading to depolarization and cell death. In brain injury, this process can be averted if the mitochondrial swelling is addressed immediately. This indicates an opportunity to introduce mitoprotective therapies in a similar vein in articular cartilage. To better understand chondrocyte fate and investigate the process of mitochondrial depolarization after strain, an understanding of the Ca2+ signaling during mechanical stimulation is required. We predict that chondrocytes in different regions of cartilage will show different Ca2+ signal responses, in relation to the depth dependent compressive and shear properties of the tissue. In order to test this prediction, we compare changes in Ca2+ signaling under various magnitudes of compressive and shear strain, and compare the signaling response with previous work that determined impact-induced strain thresholds for mitochondrial dysfunction and chondrocyte death. Methods: Explants of articular cartilage were sterilely dissected from the femoral condyles of neonatal calves. The explants were cultured in a solution of DMEM and Antibiotic-Antimycotic for 24 hours. After removal from culture, the explants were vertically bisected into hemicylinders. They were stained for Ca2+ ions in 5μM Fluo-8 AM for one hour at 37° C, and rinsed in pure DMEM three times for ten minutes each to remove excess stain. The bisected samples were affixed to a custom-built Tissue Deformation Imaging Stage (TDIS) that can apply compressive and shear strain while directly imaging the tissue on a Zeiss LSM 510 inverted confocal microscope [Fig. 1]. Each sample was subjected to compression and shear. Recordings were made for 1000s while uncompressed, immediately following compression, post-relaxation, and immediately following shear. From these videos we were able to extract cell locations as a function of time through a centroid tracking algorithm [Fig. 2]. We extracted location and intensity information for all cells framed within a 636× 636μm frame (∼1000 cells per video), and identified cells showing Ca2+ transients. Local strain was calculated using a 2D digital image correlation program and the intensity curve of each cell was matched with the corresponding local strain. Results: Cell tracking showed different families of curves in Ca2+ signal intensity for cells located in the superficial (0 to 200μm depth) and middle (> 200μm depth) regions [Fig. 3], as well as multiple different families of cell signals present for uncompressed, compressed, and sheared samples [Fig. 4]. Increased proportion of cells showing Ca2+signaling was observed at greater compressive strain and in comparison with unstrained samples, indicating a threshold at which abnormal Ca2+ signaling occurs. Conclusions: The development of a technique to track a large number of cells in a non-equilibrium state is an improvement upon previous methods. This method allows for the study of transient signals despite changes in location and brightness, allows for large-scale tracking of cells for population statistics and provides the basis to directly study the relationship between Ca2+ signaling and mitochondrial response. The observation of differences in cell Ca2+ signaling between the surface and middle zones before any tissue strain was applied reinforces the idea that the superficial layer has a protective role for articular cartilage. This is in agreement with previous work showing that removal of the superficial layer allowed strain to penetrate deeper into the tissue. The identification of strain thresholds for increased Ca2+ signaling aligns with previous work showing strain thresholds for mitochondrial depolarization and cell death, further promoting the role that Ca2+ has in the cellular response pathway to strain.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)