A Standardized Protocol for the Safe Retrieval of Infectious Postmortem Human Brain for Studying Whole-Brain Pathology.

ABSTRACT: We describe a safe and standardized perfusion protocol for studying brain pathology in high-risk autopsies using a custom-designed low-cost infection containment chamber and high-resolution histology. The output quality was studied using the histological data from the whole cerebellum and brain stem processed using a high-resolution cryohistology pipeline at 0.5 µm per pixel, in-plane resolution with serial sections at 20-µm thickness. To understand the pathophysiology of highly infectious diseases, it is necessary to have a safe and cost-effective method of performing high-risk autopsies and a standardized perfusion protocol for preparing high-quality tissues. Using the low-cost infection containment chamber, we detail the cranial autopsy protocol and ex situ perfusion-fixation of 4 highly infectious adult human brains. The digitized high-resolution histology images of the Nissl-stained series reveal that most of the sections were free of processing artifacts, such as fixation damage, freezing artifacts, and osmotic shock, at the macrocellular and microcellular level. The quality of our protocol was also tested with the highly sensitive immunohistochemistry staining for specific protein markers. Our protocol provides a safe and effective method in high-risk autopsies that allows for the evaluation of pathogen-host interaction, the underlying pathophysiology, and the extent of the infection across the whole brain at microscopic resolutions.

Dermatological Aspects of Nursing Oncology: Meaningful Observations Ensuring Better Quality of Life.

Modern cancer management has changed over the period of time and now shifted to multidisciplinary care approach to ensure a better quality of life (QOL) of the surfing patients. Every form of cancer treatment has side effects and affects the QOL. Many of the side effects have been discussed in detail because of the need for timely interventions to prevent the consequences of the side effects. Dermatological adverse events due to cancer treatment are important but most commonly ignored in our clinical practice. Nursing staffs have a critical role in the early identification of such events and by briefing and training of the nursing staff in the identification of adverse events which can aid in the prevention of complications. As dermatologists may not be available round the clock, nursing staff are looking after the patients round the clock can prove very vital in screening cutaneous AE and adequately setting up referrals to aid early recognition and treatment of not only mild but also potentially life-threatening complications. The nursing staff, which is a cadre of health caregivers that are intimately involved in cancer care, can be trained to identify timely, skin-related adverse events. A literature search of scientific publications was done using the electronic databases PubMed, Science Direct, Cochrane Library, and Google Scholar. The search included terms 'Adverse events (AEs) post-chemotherapy,' 'AE post-radiotherapy,' 'AE post-immunotherapy,' 'AE post-hormonal therapy for cancer' and 'AE post-cancer surgery.' Data obtained from these studies and case reports were compiled and interpreted to prepare this review. This review focuses on various ways in which skin can be involved adversely as a part of cancer management and their classic and tell-tale signs to help the nurses in their better and quicker identification so that dermatologists are timely intimated and the treatment can be instituted to improve the patient's QOL.

A CRISPR/Cas9 based polymeric nanoparticles to treat/inhibit microbial infections.

The latest breakthrough towards the adequate and decisive methods of gene editing tools provided by CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR Associated System), has been repurposed into a tool for genetically engineering eukaryotic cells and now considered as the major innovation in gene-related disorders. Nanotechnology has provided an alternate way to overcome the conventional problems where methods to deliver therapeutic agents have failed. The use of nanotechnology has the potential to safe-side the CRISPR/Cas9 components delivery by using customized polymeric nanoparticles for safety and efficacy. The pairing of two (CRISPR/Cas9 and nanotechnology) has the potential for opening new avenues in therapeutic use. In this review, we will discuss the most recent advances in developing nanoparticle-based CRISPR/Cas9 gene editing cargo delivery with a focus on several polymeric nanoparticles including fabrication proposals to combat microbial infections.

H2S-stimulated bioenergetics in chicken erythrocytes and the underlying mechanism.

The production of H2S and its effect on bioenergetics in mammalian cells may be evolutionarily preserved. Erythrocytes of birds, but not those of mammals, have a nucleus and mitochondria. In the present study, we report the endogenous production of H2S in chicken erythrocytes, which was mainly catalyzed by 3-mercaptopyruvate sulfur transferase (MST). ATP content of erythrocytes was increased by MST-generated endogenous H2S under normoxic, but not hypoxic, conditions. NaHS, a H2S salt, increased ATP content under normoxic, but not hypoxic, conditions. ATP contents in the absence or presence of NaHS were eliminated by different inhibitors for mitochondrial electron transport chain in chicken erythrocytes. Succinate and glutamine, but not glucose, increased ATP content. NaHS treatment similarly increased ATP content in the presence of glucose, glutamine, or succinate, respectively. Furthermore, the expression and activity of sulfide:quinone oxidoreductase were enhanced by NaHS. The structural integrity of chicken erythrocytes was largely maintained during 2-wk NaHS treatment in vitro, whereas most of the erythrocytes without NaHS treatment were lysed. In conclusion, H2S may regulate cellular bioenergetics as well as cell survival of chicken erythrocytes, in which the functionality of the electron transport chain is involved. H2S may have different regulatory roles and mechanisms in bioenergetics of mammalian and bird cells.

A nanovaccine formulation of Chlamydia recombinant MOMP encapsulated in PLGA 85:15 nanoparticles augments CD4+ effector (CD44high CD62Llow) and memory (CD44high CD62Lhigh) T-cells in immunized mice.

Vaccine developmental strategies are utilizing antigens encapsulated in biodegradable polymeric nanoparticles. Here, we developed a Chlamydia nanovaccine (PLGA-rMOMP) by encapsulating its recombinant major outer membrane protein (rMOMP) in the extended-releasing and self-adjuvanting PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. PLGA-rMOMP was small (nanometer size), round and smooth, thermally stable, and exhibited a sustained release of rMOMP. Stimulation of mouse primary dendritic cells (DCs) with PLGA-rMOMP augmented endosome processing, induced Th1 cytokines (IL-6 and IL-12p40), and expression of MHC-II and co-stimulatory (CD40, CD80, and CD86) molecules. BALB/c mice immunized with PLGA-rMOMP produced enhanced CD4+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) phenotypes and functional antigen-specific serum IgG antibodies. In vivo biodistribution of PLGA-rMOMP revealed its localization within lymph nodes, suggesting migration from the injection site via DCs. Our data provide evidence that the PLGA (85:15) nanovaccine activates DCs and augments Chlamydia-specific rMOMP adaptive immune responses that are worthy of efficacy testing.

Double vascularized omentum lymphatic transplant (VOLT) for the treatment of lymphedema.

BACKGROUND AND OBJECTIVES: Orthotopic vascularized lymph node transplant has been successfully used to treat lymphedema. A second, heterotopic lymph node transplant in the distal extremity may provide further improvement. The vascularized omentum lymphatic transplant (VOLT) provides adequate tissue for two simultaneous flap transfers to one limb. The purpose of this study was to review our experience with this technique. METHODS: We conducted a retrospective study of patients who underwent VOLT, with a subgroup analysis of patients who underwent double VOLT. Technical aspects of the procedure, complications, and early outcomes were reviewed. RESULTS: From May 2015 to August 2017, 54 VOLTs were performed in 38 patients, of whom 16 received double VOLT. Among patients in the double VOLT group with postoperative imaging at 1 year, uptake into the transplanted omentum was seen in three of six (50%) patients on lymphoscintigraphy and in one of five (20%) patients on indocyanine green lymphangiography. One patient (3.1%) in the double VOLT group required a return to the operating room. There were no donor site complications in the double VOLT group. The overall complication rate was 15.8%. CONCLUSIONS: Double VOLT to the mid-level and proximal extremity is a safe and viable option.

Leishmania donovani molecules recognized by sera of filaria infected host facilitate filarial infection.

We earlier found that F6 fraction of human filaria Brugia malayi cross-reacted with sera of Leishmania donovani infected hamsters and immunization with F6 inhibited both filarial and leishmanial infections. In the present study, we identified a 52.9-93.6 kDa fraction (Ld1) of L. donovani that cross-reacted with sera of B. malayi infected animals and investigated effect of Ld1 on filarial infection. Immunization of BALB/c mice with Ld1 facilitated B. malayi infection with remarkable increase in parasite burden. Facilitation of filarial infection was associated with downregulated cell proliferation, IL-5, IL-13, IFN-γ, TNF-α, and IL-2 levels and upregulated IL-4 and TGF-ß. Ld1 exposure also suppressed MHC class-I, MHC class-II, and FcεR1 expression, and phagocytosis in naive mouse macrophages, and CD4+, CD8+, and CD19+ cell population in mouse spleen. Two-dimensional electrophoresis and matrix-assisted laser desorption ionization-time of flight-mass spectrometry revealed eight proteins in Ld1: putative heat shock protein (HSP) 70-related protein 1, HSP70 mitochondrial precursor, alanine aminotransferase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, protein disulfide isomerase, putative ATPase beta subunit, trypanothione reductase, and a hypothetical protein. HSP70 protein mitochondrial precursor and trypanothione reductase showed homology with Trypanosoma cruzi and L. donovani, respectively, and the rest 6 proteins including hypothetical protein bear homology with L. infantum. In conclusion, the present study for the first time shows that immunization with filarial cross-reactive Ld1 fraction of L. donovani facilitates filarial infection by modulating Th1 and Th2 responses. Ld1 molecules may therefore facilitate filarial infection in filaria-leishmania co-infection.

Tibialis Anterior Muscle Hernia: A Case of Chronic, Dull Pain and Swelling in Leg Diagnosed by Dynamic Ultrasonography.

BACKGROUND: Symptomatic muscle hernias are not uncommon in the lower extremities and are a rare cause of chronic leg pain. They are most commonly seen in the tibialis anterior muscle, occurring through fascial defects, usually after trauma. There are about 200 cases of muscle hernias described in the literature. The diagnosis is challenging as most of the patients present with non-specific chronic leg pain. Dynamic muscle ultrasonography at rest and at stress is often used for the diagnosis. CASE REPORT: We describe a case of tibialis anterior muscle hernia presenting with persistent dull pain and swelling along the anterior aspect of the leg on straining the leg muscles. Dynamic ultrasonography was performed, which showed a defect in the fascial sheath of the muscle through which the tibialis anterior muscle herniated and produced a focal bulge along the anterior aspect of the leg. Based upon physical examination and dynamic ultrasonographic findings, a diagnosis of tibialis anterior muscle hernia was made. CONCLUSIONS: Tibialis anterior muscle hernia is a rare diagnosis and should be included in the differential diagnosis in a patient with chronic leg pain and swelling. Dynamic ultrasound is crucial in confirming the diagnosis and should be done on straining the muscles of the affected limb.

Comparison of Laser Doppler and Laser-Assisted Indocyanine Green Angiography Prediction of Flap Survival in a Novel Modification of the McFarlane Flap.

BACKGROUND: The McFarlane rat ischemic dorsal skin flap model has been commonly used for clinical vector studies, as well as the testing of noninvasive diagnostics. However, variability of this model secondary to flap contact with the wound bed has led many to question its validity. Here we present a novel modification to the McFarlane skin flap using sterile silicone. We also use this model to test the prognostic efficacy of laser-assisted indocyanine green (ICG) angiography and laser Doppler imaging (LDI). METHODOLOGY: A 3 × 9-cm dorsal skin flap with a cranially based pedicle was created, centered 1 cm distal to the scapulae. The flap was undermined, and in one of the 2 groups, a sterile silicone sheet was placed onto the wound bed. All flaps were then reapproximated with sutures 1-cm intervals. Clinical assessment and perfusion imaging was performed immediately postoperative, and at 24, 48, and 72 hours postsurgery. Postoperative day 7 clinical assessment was obtained before euthanasia. RESULTS: A comparative study using silicone blocked versus unblocked models (n = 6 per group) showed that, clinically, both models had equivalent flap survival [8.5 (0.913) vs 9.5 (1.01) cm]. However, a statistically significant increase in perfusion in the mid-third of unblocked models was observed on POD3 [20.28% (2.7%) vs blocked 13.45% (2.5%), P < 0.05], with a similar increase in the distal third on POD7 [18.73% (2.064%) vs 10.91% (4.19%), P < 0.05]. A prognostic study comparing LDI and ICG angiography prediction of POD7 survival at early time points (n = 10) found that LDI underpredicted flap survival at early time points [84.2% (12.03%) on POD0, 87.35% (16.11%) on POD1]. In contrast, ICG was more proficient [100.1% (10.1%) on POD0]. CONCLUSIONS: We present a modification of the McFarlane skin flap model that results in similar clinical results, but with a noted reduction in perfusion inconsistencies noted in unblocked models. The ICG angiography is superior to LDI in predicting POD7 flap necrosis within the first 48 hours postinjury. Future work will focus on histologic validation of our model, and vector efficacy testing.

Effect of scaling on serum high-sensitivity C-reactive protein levels and periodontal parameters in systemically healthy women of reproductive age group with gingivitis.

OBJECTIVE: Varying levels of sex hormones across the menstrual cycle in young systemically healthy females may alter tissue responses to plaque, resulting in increased gingival inflammation. Also, higher severity and prevalence of gingivitis has been demonstrated in adult females than males, attributed to hormonal changes. Further, literature reported that Gingivitis raises the levels of systemic inflammatory markers such as C Reactive Protein. This interventional trial aimed to evaluate the effect of supragingival scaling on serum highsensitivity C-reactive protein (hsCRP) levels along with periodontal parameters in systemically healthy women of reproductive age group with natural gingivitis. MATERIAL AND METHODS: 57 women of reproductive age were enrolled into two groups. Test Group (n=30) comprised of systemically healthy women with gingivitis who received supragingival scaling. Control Group (n=27) included systemically and periodontally healthy females. Periodontal parameters [gingival index (GI), plaque index (PI), pocket probing depth (PPD), bleeding on probing (BOP)], and serum hsCRP levels were recorded at baseline for both the groups. Follow up of Test Group participants was done at 3 and 6 months. RESULTS: Serum hsCRP and periodontal parameters were significantly higher in Test Group than control group at baseline which decreased significantly after treatment in Test Group at 6 months follow up (p≤0.05). GI, BOP and hsCRP in Test Group at 6 months were reduced up to the baseline levels of systemically and periodontally healthy females. CONCLUSION: Treatment of gingival inflammation can help in lowering the systemic and local inflammation up to the levels of systemically and periodontally healthy females.

Platelet-Rich Fibrin (PRF) and its Application in Dentistry: A Literature Review.

Platelet-rich fibrin (PRF) is a biocompatible fibrin matrix that acts as a resorbable membrane, collecting platelet cytokines, growth factors, and cells and releasing them. It is easily extracted from blood. Many dentists use PRF, a concentrated growth factor solution, to treat various lesions and regenerate tooth and oral tissues. This research examines the pros, cons, and suggestions of using PRF in dentistry.

Effects of prime-boost strategies on the protective efficacy and immunogenicity of a PLGA (85:15)-encapsulated Chlamydia recombinant MOMP nanovaccine.

To begin to optimize the immunization routes for our reported PLGA-rMOMP nanovaccine [PLGA-encapsulated Chlamydia muridarum (Cm) recombinant major outer membrane protein (rMOMP)], we compared two prime-boost immunization strategies [subcutaneous (SC) and intramuscular (IM-p) prime routes followed by two SC-boosts)] to evaluate the nanovaccine-induced protective efficacy and immunogenicity in female BALB/c mice. Our results showed that mice immunized via the SC and IM-p routes were protected against a Cm genital challenge by a reduction in bacterial burden and with fewer bacteria in the SC mice. Protection of mice correlated with rMOMP-specific Th1 (IL-2 and IFN-γ) and not Th2 (IL-4, IL-9, and IL-13) cytokines, and CD4+ memory (CD44highCD62Lhigh) T-cells, especially in the SC mice. We also observed higher levels of IL-1α, IL-6, IL-17, CCL-2, and G-CSF in SC-immunized mice. Notably, an increase of cytokines/chemokines was seen after the challenge in the SC, IM-p, and control mice (rMOMP and PBS), suggesting a Cm stimulation. In parallel, rMOMP-specific Th1 (IgG2a and IgG2b) and Th2 (IgG1) serum, mucosal, serum avidity, and neutralizing antibodies were more elevated in SC than in IM-p mice. Overall, the homologous SC prime-boost immunization of mice induced enhanced cellular and antibody responses with better protection against a genital challenge compared to the heterologous IM-p.

Comparison of colorimetric, spectroscopic and electrochemical techniques for quantification of hydrogen sulfide.

Background: Hydrogen sulfide (H2S), an endogenous gasotransmitter, has potential applications in several conditions. However, its quantification in simulated physiological solutions is a major challenge due to its gaseous nature and other physicochemical properties. Aim: This study was designed to compare four commonly used H2S detection and quantification methods in aqueous solutions. Methods: The four techniques compared were one colorimetric, one chromatographic and two electrochemical methods. Results: Colorimetric and chromatographic methods quantified H2S in millimolar and micromole ranges, respectively. The electrochemical methods quantified H2S in the nanomole and picomole ranges and were less time-consuming. Conclusion: The H2S quantification method should be selected based on the specific requirements of a research project in terms of sensitivity, response time and cost-effectiveness.

Systematic development of immunohistochemistry protocol for large cryosections-specific to non-perfused fetal brain.

BACKGROUND: Immunohistochemistry (IHC) is an important technique in understanding the expression of neurochemical molecules in the developing human brain. Despite its routine application in the research and clinical setup, the IHC protocol specific for soft fragile fetal brains that are fixed using the non-perfusion method is still limited in studying the whole brain. NEW METHOD: This study shows that the IHC protocols, using a chromogenic detection system, used in animals and adult humans are not optimal in the fetal brains. We have optimized key steps from Antigen retrieval (AR) to chromogen visualization for formalin-fixed whole-brain cryosections (20 µm) mounted on glass slides. RESULTS: We show the results from six validated, commonly used antibodies to study the fetal brain. We achieved optimal antigen retrieval with 0.1 M Boric Acid, pH 9.0 at 70°C for 20 minutes. We also present the optimal incubation duration and temperature for protein blocking and the primary antibody that results in specific antigen labeling with minimal tissue damage. COMPARISON WITH EXISTING METHODS: The IHC protocol commonly used for adult human and animal brains results in significant tissue damage in the fetal brains with little or suboptimal antigen expression. Our new method with important modifications including the temperature, duration, and choice of the alkaline buffer for AR addresses these pitfalls and provides high-quality results. CONCLUSION: The optimized IHC protocol for the developing human brain (13-22 GW) provides a high-quality, repeatable, and reliable method for studying chemoarchitecture in neurotypical and pathological conditions across different gestational ages.

Histological characterization and development of mesial surface sulci in the human brain at 13-15 gestational weeks through high-resolution histology.

Cellular-level anatomical data from early fetal brain are sparse yet critical to the understanding of neurodevelopmental disorders. We characterize the organization of the human cerebral cortex between 13 and 15 gestational weeks using high-resolution whole-brain histological data sets complimented with multimodal imaging. We observed the heretofore underrecognized, reproducible presence of infolds on the mesial surface of the cerebral hemispheres. Of note at this stage, when most of the cerebrum is occupied by lateral ventricles and the corpus callosum is incompletely developed, we postulate that these mesial infolds represent the primordial stage of cingulate, callosal, and calcarine sulci, features of mesial cortical development. Our observations are based on the multimodal approach and further include histological three-dimensional reconstruction that highlights the importance of the plane of sectioning. We describe the laminar organization of the developing cortical mantle, including these infolds from the marginal to ventricular zone, with Nissl, hematoxylin and eosin, and glial fibrillary acidic protein (GFAP) immunohistochemistry. Despite the absence of major sulci on the dorsal surface, the boundaries among the orbital, frontal, parietal, and occipital cortex were very well demarcated, primarily by the cytoarchitecture differences in the organization of the subplate (SP) and intermediate zone (IZ) in these locations. The parietal region has the thickest cortical plate (CP), SP, and IZ, whereas the orbital region shows the thinnest CP and reveals an extra cell-sparse layer above the bilaminar SP. The subcortical structures show intensely GFAP-immunolabeled soma, absent in the cerebral mantle. Our findings establish a normative neurodevelopment baseline at the early stage.

Glycyrrhetinic acid and its analogs: a new class of antifilarial agents.

Although a number of chemicals have been isolated from Glycyrrhiza glabra, only a few have been evaluated for their biological significance. As part of our drug discovery program for antifilarial agents from Indian medicinal plants, the roots of G. glabra were chemically investigated, which resulted in the isolation and characterization of an antifilarial agent, glycyrrhetinic acid (GA, 1a) effective against microfilariae (mf) in vitro (LC100: 12.5 µM; IC50: 1.20 µM), but was inactive against adult worms. Further, GA (1a) was converted into six analogs (2a-7a) and their antifilarial potential was evaluated by studying in vitro motility and MTT reduction assays employing mf and adult worms of Brugia malayi. The results showed that out of six GA analogs, the benzyl amide analog (6a) killed adults and mf at 25 and 50 µM concentration, respectively, and inhibited 49% MTT reduction potential of the adult parasites. The IC50 values were found to be 8.8 and 2.2 µM for adults and mf, respectively. The SI of the compound was >60. On the other hand the octylamide analog (7a) required much higher concentration to adversely affect the parasites. Finally, both active amide analogs (6a and 7a) were in vivo evaluated using B. malayi-jird model, which showed that analog 6a possesses promising macrofilaricidal activity at 100mg/kg, s.c. ×5 days and around 40% of the treated animals showed calcified masses of worm fragments in peritoneal cavity of the animals. To the best of our knowledge this is the first ever report on the antifilarial potential of GA analogs. Further work on optimization of the antifilarial lead is under progress.

Conv-CapsNet: capsule based network for COVID-19 detection through X-Ray scans.

Coronavirus, a virus that spread worldwide rapidly and was eventually declared a pandemic. The rapid spread made it essential to detect Coronavirus infected people to control the further spread. Recent studies show that radiological images such as X-Rays and CT scans provide essential information in detecting infection using deep learning models. This paper proposes a shallow architecture based on Capsule Networks with convolutional layers to detect COVID-19 infected persons. The proposed method combines the ability of the capsule network to understand spatial information with convolutional layers for efficient feature extraction. Due to the model's shallow architecture, it has 23M parameters to train and requires fewer training samples. The proposed system is fast and robust and correctly classifies the X-Ray images into three classes, i.e. COVID-19, No Findings, and Viral Pneumonia. Experimental results on the X-Ray dataset show that our model performs well despite having fewer samples for the training and achieved an average accuracy of 96.47% for multi-class and 97.69% for binary classification on 5-fold cross-validation. The proposed model would be useful to researchers and medical professionals for assistance and prognosis for COVID-19 infected patients.

Wide field block face imaging using deep ultraviolet induced autofluorescence of the human brain.

BACKGROUND: Imaging large volume human brains at cellular resolution involve histological methods that cause structural changes. A reference point prior to sectioning is needed to quantify these changes and is achieved by serial block face imaging (BFI) methods that have been applied to small volume tissue (∼1 cm3). NEW METHOD: We have developed a BFI uniquely designed for large volume tissues (∼1300 cm3) with a very large field of view (20 × 20 cm) at a resolution of 70 µm/pixel under deep ultraviolet (UV-C) illumination which highlights key features. RESULTS: The UV-C imaging ensures high contrast imaging of the brain tissue and highlights salient features of the brain. The system is designed to provide uniform and stable illumination across the entire surface area of the tissue and to work at low temperatures, which are required during cryosectioning. Most importantly, it has been designed to maintain its optical focus over the large depth of tissue and over long periods of time, without readjustments. The BFI was installed within a cryomacrotome, and was used to image a large cryoblock of an adult human cerebellum and brainstem (∼6 cm depth resulting in 2995 serial images) with precise optical focus and no loss during continuous serial acquisition. COMPARISON WITH EXISTING METHOD(S): The deep UV-C induced BFI highlights several large fibre tracts within the brain including the cerebellar peduncles, and the corticospinal tract providing important advantage over white light BFI. CONCLUSIONS: The 3D reconstructed serial BFI images can assist in the registration and alignment of the microscopic high-resolution histological tissue sections.

A technology platform for standardized cryoprotection and freezing of large-volume brain tissues for high-resolution histology.

Understanding and mapping the human connectome is a long-standing endeavor of neuroscience, yet the significant challenges associated with the large size of the human brain during cryosectioning remain unsolved. While smaller brains, such as rodents and marmosets, have been the focus of previous connectomics projects, the processing of the larger human brain requires significant technological advancements. This study addresses the problem of freezing large brains in aligned neuroanatomical coordinates with minimal tissue damage, facilitating large-scale distortion-free cryosectioning. We report the most effective and stable freezing technique utilizing an appropriate choice of cryoprotection and leveraging engineering tools such as brain master patterns, custom-designed molds, and a continuous temperature monitoring system. This standardized approach to freezing enables high-quality, distortion-free histology, allowing researchers worldwide to explore the complexities of the human brain at a cellular level. Our approach combines neuroscience and engineering technologies to address this long-standing challenge with limited resources, enhancing accessibility of large-scale scientific endeavors beyond developed countries, promoting diverse approaches, and fostering collaborations.

A synthetic S6 segment derived from KvAP channel self-assembles, permeabilizes lipid vesicles, and exhibits ion channel activity in bilayer lipid membrane.

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.