Herb Extracellular Vesicle-Chitosan-PEGylated Graphene Oxide Conjugate Delivers Estrogen Receptor α Targeting siRNA to Breast Cancer Cells.

Herb-based extracellular vesicles (EV), inherently replete with bioactive proteins, RNA, lipids, and other medicinal compounds, are noncytotoxic and uniquely capable of cellular delivery to meet the ever-stringent challenges of ongoing clinical applications. EVs are abundant in nature, affordable, and scalable, but they are also incredibly fragile and stuffed with many biomolecules. To address the low drug binding abilities and poor stability of EVs, we demonstrated herb-based EVs (isolated from neem, mint, and curry leaves) conjugated with chitosan (CS) and PEGylated graphene oxide (GP) that led to their transformation into robust and efficient vectors. The designed conjugates successfully delivered estrogen receptor α (ERα1)-targeting siRNA to breast cancer MCF7 cells. Our data revealed that neem-based EV-CS-GP conjugates were most efficient in cellular siRNA delivery, which could be attributed to hyaluronic acid-mediated recognition of neem EVs by MCF7 cells via CD44 receptors. Our approach shows a futuristic direction in designing clinically viable, sustainable, nontoxic EV-based vehicles that can deliver a variety of functional siRNA cargos.

Probing Aberrantly Glycosylated Mucin 1 in Breast Cancer Extracellular Vesicles.

Aberrantly glycosylated mucin 1 is a critical prognostic biomarker in breast epithelial cancers. Hypoglycosylated mucin 1 coats the surface of the cancer cells, where O-glycans are predominantly linked via an N-acetylgalactosamine moiety (GalNAc). Cancer cell-derived extracellular vesicles (EVs) carry biomarkers from parent cancer cells to the recipient cells and, therefore, could potentially be leveraged for diagnostics and noninvasive disease monitoring. We devised a label-free approach for identifying glycoprotein mucin 1 overexpression on breast cancer EVs. While exploring a plethora of biochemical (enzyme-linked immunosorbent assay, flow cytometry, and SDS-PAGE) and label-free biophysical techniques (circular dichroism and infrared spectroscopy (IR)) along with multivariate analysis, we discovered that mucin 1 is significantly overexpressed in breast cancer EVs and aberrant glycosylation in mucin 1 could be critically addressed using IR and multivariate analysis targeting the GalNAc sugar. This approach emerges as a convenient and comprehensive method of distinguishing cancer EVs from normal samples and holds potential for nonintrusive breast cancer liquid biopsy screening.

Label-Free Physical-Analytical Techniques Reveal Epigenetic Modifications of Breast Cancer Chromosomes.

Epigenetic dysregulation including DNA methylation and histone modifications is being increasingly recognized as a promising biomarker for the diagnosis and prognosis of cancer. Herein, we devised a label-free analytical toolbox comprising IR, UV-vis, CD spectroscopy, and cyclic voltammetry, which is capable to differentiate significantly hyper-methylated breast cancer chromosomes from the normal breast epithelial counterparts.

Seroprevalence of SARS-CoV-2 antibodies among Forcibly Displaced Myanmar Nationals in Cox's Bazar, Bangladesh 2020: a population-based cross-sectional study.

OBJECTIVES: The study aimed to determine the seroprevalence, the fraction of asymptomatic infections, and risk factors of SARS-CoV-2 infections among the Forcibly Displaced Myanmar Nationals (FDMNs). DESIGN: It was a population-based two-stage cross-sectional study at the level of households. SETTING: The study was conducted in December 2020 among household members of the FDMN population living in the 34 camps of Ukhia and Teknaf Upazila of Cox's Bazar district in Bangladesh. PARTICIPANTS: Among 860 697 FDMNs residing in 187 517 households, 3446 were recruited for the study. One individual aged 1 year or older was randomly selected from each targeted household. PRIMARY AND SECONDARY OUTCOME MEASURES: Blood samples from respondents were tested for total antibodies for SARS-CoV-2 using Wantai ELISA kits, and later positive samples were validated by Kantaro kits. RESULTS: More than half (55.3%) of the respondents were females, aged 23 median (IQR 14-35) years and more than half (58.4%) had no formal education. Overall, 2090 of 3446 study participants tested positive for SARS-CoV-2 antibody. The weighted and test adjusted seroprevalence (95% CI) was 48.3% (45.3% to 51.4%), which did not differ by the sexes. Children (aged 1-17 years) had a significantly lower seroprevalence 38.6% (95% CI 33.8% to 43.4%) compared with adults (58.1%, 95% CI 55.2% to 61.1%). Almost half (45.7%, 95% CI 41.9% to 49.5%) of seropositive individuals reported no relevant symptoms since March 2020. Antibody seroprevalence was higher in those with any comorbidity (57.8%, 95% CI 50.4% to 64.5%) than those without (47.2%, 95% CI 43.9% to 50.4%). Multivariate logistic regression analysis of all subjects identified increasing age and education as risk factors for seropositivity. In children (≤17 years), only age was significantly associated with the infection. CONCLUSIONS: In December 2020, about half of the FDMNs had antibodies against SARS-CoV-2, including those who reported no history of symptoms. Periodic serosurveys are necessary to recommend appropriate public health measures to limit transmission.

A Mechanoelastic Glimpse on Hyaluronan-Coated Extracellular Vesicles.

Cancer cells secrete extracellular vesicles (EVs) covered with a carbohydrate polymer, hyaluronan (HA), linked to tumor malignancy. Herein, we have unravelled the contour lengths of HA on a single cancer cell-derived EV surface using single-molecule force spectroscopy (SMFS), which divulges the presence of low molecular weight HA (LMW-HA < 200 kDa). We also discovered that these LMW-HA-EVs are significantly more elastic than the normal cell-derived EVs. This intrinsic elasticity of cancer EVs could be directly allied to the LMW-HA abundance and associated labile water network on EV surface as revealed by correlative SMFS, hydration dynamics with fluorescence spectroscopy, and molecular dynamics simulations. This method emerges as a molecular biosensor of the cancer microenvironment.

Contralateral Acute Extradural Hematoma Following Decompressive Craniectomy for Subdural Hematoma Evacuation: A Rare Complication and a Short Literature Review.

Subdural hematoma (SDH) is a common intracranial lesion seen in severe traumatic brain injury (TBI). The development of contralateral delayed extradural hematoma (EDH) after surgery is well described. But limited literature available about immediate contralateral EDH after subdural hematoma (SDH) evacuation. We report a case of a young adult who developed contralateral EDH following decompressive surgery for acute SDH.

Non-COVID surgical load of operation theatre during COVID-19 pandemic in Armed Forces tertiary care centres: "Snapshot of two timelines".

Background: The global COVID-19 pandemic has led to major changes in the surgical caseloads in the operation theatres across the world. Elective surgeries have been curtailed to a great extent and the number of emergency surgeries has risen. We conducted a study to assess the changes in the surgical caseloads in two tertiary care hospitals in India during the pandemic and to assess the changes in anaesthesia techniques used. Methods: A multicentre retrospective observational study conducted at two tertiary care centres located 1500 kms apart. Hospital A in a city with high incidence of COVID-19 and Hospital B with a lighter load of COVID-19 cases. Record of all major surgeries carried out between 01Jul 2020 and 31 Dec 2020 were compared with the surgeries carried out between 01 Jul 2019 and 31 Dec 2019. Results: There was a decrease of almost 53% in elective surgical workload in hospital A and 71% in hospital B. The decrease in the cases was not similar across all specialities. There was a significant increase in the percentage of emergency surgeries from 11.1% to 24% in hospital A and from 22.1% to 29.8% in hospital B in the year 2020. A statistically significant increase in regional anaesthesia and neuraxial blockade was noted in the year 2020 in both the hospitals. Conclusion: During the COVID-19 pandemic, the number of elective surgical cases has significantly decreased in specialities like ophthalmology whereas there was no change in the surgical workload in specialities like obstetrics, oncology, orthopaedics, and neurosurgery. There was significant increase in the use of regional and neuraxial techniques of anaesthesia.

Relationship of Computed Tomography Severity Score With Patient Characteristics and Survival in Hypoxemic COVID-19 Patients.

Background Computed tomography (CT) scans and CT severity scores (CTSS) are widely used to assess the severity and prognosis in coronavirus disease 2019 (COVID-19). CTSS has performed well as a predictor in differentiating severe from non-severe cases. However, it is not known if CTSS performs similarly in hospitalized severe cases with hypoxia at admission. Methods We conducted a retrospective comparative study at a COVID-care center from Western India between 25th April and 31st May 2021, enrolling all consecutive severe COVID-19 patients with hypoxemia (peripheral oxygen saturation < 94%). Neutrophil-lymphocyte ratio (NLR), C-reactive protein (CRP), interleukin-6 (IL-6), lactate dehydrogenase (LDH), D-dimer, ferritin, and CT thorax were done within 24h of admission before being initiated on any anti-COVID-19 therapy. CTSS was calculated by visual assessment and categorized into three severity categories and was correlated with laboratory markers and overall survival (OS). Statistical analysis was done using John's Macintosh Project (JMP) 15.0.0 ver. 3.0.0 (Cary, North Carolina). Results The median age of the study population (n-298) was 59 years (24-95) with a male preponderance (61.41%, n=183). The 15 and 30-day survivals were 67.64% and 59.90%, respectively. CTSS did not correlate with age, gender, time from vaccination, symptoms, or comorbidities but had a significant though weak correlation with LDH (p=0.009), D-dimer (p=0.006), and NLR (p=0.019). Comparing demographic and laboratory aspects using CT severity categories, only NLR (p=0.0146) and D-dimer (p=0.0006) had significant differences. The 15d-OS of mild, moderate, and severe CT categories were 88.62%, 70.39%, and 52.62%, respectively, while 30d-OS of three categories were 59.08%, 63.96%, and 49.12%, respectively. Conclusion Among hospitalized severe COVID-19 patients with hypoxemia at admission, CT severity categories correlate well with outcomes but not inflammatory markers at admission.

Headache of Wearing PPE; A Survey for Neurological Symptoms with PPE amongst Health Care Workers.

Background: The COVID-19 pandemic has put the entire medical fraternity into a very challenging and demanding situation. Along with always being at the risk of COVID infection, healthcare workers (HCWs) are also facing neurological problems due to long working hours in personal protective equipment (PPE). These symptoms and their characteristics need to be observed and studied in-depth to understand the problems experienced by HCWs and to design new solutions to overcome such problems. Objectives: This study intends to evaluate the various neurological manifestations among the HCWs wearing PPE for prolonged periods. Materials and Methods: We conducted a questionnaire-based cross-sectional study at a Covid care center from western India from April 20 to June 01, 2021 by using a self-administered web-based questionnaire. A total of 256 HCWs were surveyed. The de-identified data were analyzed using JMP 15.0.0. Results: Among a total of 256 HCWs surveyed for this study, the majority (58.6%) were aged 24-35 years, with a male preponderance (65.62%, n = 168). Participants included doctors (41%), nurses (35%), paramedical staff (22%), and housekeeping staff (1%). The symptoms encountered among the HCWs wearing the PPE were headache, classified further as donning headache in 112 (44.98%), doffing headache in 56 (26.24%), slowed mentation in 48 (21.05%), and excessive sleepiness in 86 (38.74%), which affected their work performance. The age of the HCWs had a significant correlation with all the symptoms. Conclusion: Headache, slowed mentation, and excessive sleepiness was encountered among the HCWs wearing PPE, which depended upon the duration of PPE usage. The most common symptom was headache, which was of moderate to severe intensity.

Importance of Extracellular Vesicle Derived RNAs as Critical Colorectal Cancer Biomarkers.

Colorectal cancer typically begins from a nonmalignant polyp formation in the large intestine that, over time, develops into colorectal cancer. The growth of benign polyps can be checked if detected in the early stages of the disease. Doctors usually recommend colonoscopy to average and high-risk individuals for colorectal cancer screening. Elevated carcinoembryonic antigen (CEA) is a broadly used biomarker for colorectal cancer. The genetic and epigenetic alteration of genes such as p53, BRAF, APC, and PIK3CA is also correlated with colorectal cancer in various clinical studies. In general, tissue biopsy is most frequently used for colorectal cancer diagnosis, but the whole tumor heterogeneity cannot be accessed by this technique. Furthermore, such a highly invasive technique is not suitable for repeated testing. Recently, extracellular vesicles (EVs), lipid bilayer enclosed sacs secreted from colorectal cancer cells, are emerging as a diagnostic tool for colon cancer detection. The major advantages of using EVs for colon cancer diagnosis are (i) EVs can be isolated in a noninvasive manner from the body fluid and (ii) EV incorporated cargoes (mostly RNAs) reveal various aspects of colorectal cancer. EV-RNAs are also implicated in tumor invasion and influence the immune system for the further spread of tumors. However, due to the lack of standardized EV detection strategies, diagnostic applicability is limited. Herein, we review the recent literature on the pathobiological dependence of colorectal cancer on EV-RNAs. Further, we present the advantages of identification and characterization of EV-RNAs to explore the connection between differential expression of extracellular vesicle incorporated RNAs and colorectal cancer. How this approach may potentially translate into point of care colorectal cancer diagnostics is also discussed.

Anaesthesia in Indian Detective Fiction; The curious case of Byomkesh Bakshi.

Spinal Anesthesia; Procaine; Fictions.

Polyethylene Glycol-Mediated Fusion of Extracellular Vesicles with Cationic Liposomes for the Design of Hybrid Delivery Systems.

To realize a customizable biogenic delivery platform, herein we propose combining cell-derived extracellular vesicles (EVs) derived from breast cancer cell line MCF-7 with synthetic cationic liposomes using a fusogenic agent, polyethylene glycol (PEG). We performed a fluorescence resonance energy transfer (FRET)-based lipid-mixing assay with varying PEG 1000 concentrations (0%, 15%, and 30%) correlated with flow cytometry-based analysis and supported by dimensional analysis by dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) to validate our fusion strategy. Our data revealed that these hybrid vesicles at a particular concentration of PEG (∼15%) improved the cellular delivery efficiency of a model siRNA molecule to the EV parental breast cancer cells, MCF-7, by factors of 2 and 4 compared to the loaded liposome and EV precursors, respectively. The critical rigidity/pliability balance of the hybrid systems fused by PEG seems to be playing a pivotal role in improving their delivery capability. This approach can provide clinically viable delivery solutions using EVs.

Intriguing Biomedical Applications of Synthetic and Natural Cell-Derived Vesicles: A Comparative Overview.

The significant role of a vesicle is well recognized; however, only lately has the advancement in biomedical applications started to uncover their usefulness. Although the concept of vesicles originates from cell biology, it later transferred to chemistry and material science to develop nanoscale artificial vesicles for biomedical applications. Herein, we examine different synthetic and biological vesicles and their applications in the biomedical field in general. As our understanding of biological vesicles increases, more suitable biomimicking synthetic vesicles will be developed. The comparative discussion between synthetic and natural vesicles for biomedical applications is a relevant topic, and we envision this could enable the development of a proper approach to realize the next-generation treatment goals.

Differential flexibility leading to crucial microelastic properties of asymmetric lipid vesicles for cellular transfection: A combined spectroscopic and atomic force microscopy studies.

The role of microscopic elasticity of nano-carriers in cellular uptake is an important aspect in biomedical research. Herein we have used AFM nano-indentation force spectroscopy and Förster resonance energy transfer (FRET) measurements to probe microelastic properties of three novel cationic liposomes based on di-alkyl dihydroxy ethyl ammonium chloride based lipids having asymmetry in their hydrophobic chains (Lip1818, Lip1814 and Lip1810). AFM data reveals that symmetry in hydrophobic chains of a cationic lipid (Lip1818) imparts higher rigidity to the resulting liposomes than those based on asymmetric lipids (Lip1814 and Lip1810). The stiffness of the cationic liposomes is found to decrease with increasing asymmetry in the hydrophobic lipid chains in the order of Lip1818 > Lip1814 > lip1810. FRET measurements between Coumarin 500 (Donor) and Merocyanine 540 (Acceptor) have revealed that full width at half-maxima (hw) of the probability distribution (P(r)) of donor-acceptor distance (r), increases in an order Lip1818 < Lip1814 < Lip1810 with increasing asymmetry of the hydrophobic lipid chains. This increase in width (hw) of the donor-acceptor distance distributions is reflective of increasing flexibility of the liposomes with increasing asymmetry of their constituent lipids. Thus, the results from AFM and FRET studies are complementary to each other and indicates that an increase in asymmetry of the hydrophobic lipid chains increases elasticity and or flexibility of the corresponding liposomes. Cell biology experiments confirm that liposomal flexibility or rigidity directly influences their cellular transfection efficiency, where Lip1814 is found to be superior than the other two liposomes manifesting that a critical balance between flexibility and rigidity of the cationic liposomes is key to efficient cellular uptake. Taken together, our studies reveal how asymmetry in the molecular architecture of the hydrophobic lipid chains influences the microelastic properties of the liposomes, and hence, their cellular uptake efficiency.

Identification of Biomarker Hyaluronan on Colon Cancer Extracellular Vesicles Using Correlative AFM and Spectroscopy.

Extracellular vesicles (EVs), naturally occurring nanosized vesicles secreted from cells, are essential for intercellular communication. They carry unique biomolecules on the surface or interior that are of great interest as biomarkers for various pathological conditions such as cancer. In this work, we use high-resolution atomic force microscopy (AFM) and spectroscopy (AFS) techniques to demonstrate differences between EVs derived from colon cancer cells and colon epithelial cells at the single-vesicle level. We observe that EV populations are significantly increased in the cancer cell media compared to the normal cell EVs. We show that both EVs display an EV marker, CD9, while EVs derived from the cancer cells are slightly higher in density. Hyaluronan (HA) is a nonsulfated glycosaminoglycan linked to malignant tumor growth according to recent reports. Interestingly, at the single-vesicle level, colon cancer EVs exhibit significantly increased HA surface densities compared to the normal EVs. Spectroscopic measurements such as Fourier transform infrared (FT-IR), circular dichroism (CD), and Raman spectroscopy unequivocally support the AFM and AFS measurements. To our knowledge, it represents the first report of detecting HA-coated EVs as a potential colon cancer biomarker. Taken together, this sensitive approach will be useful in identifying biomarkers in the early stages of detection and evaluation of cancer.