Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

UNLABELLED: Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. IMPORTANCE: The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry.

The Ebola virus matrix protein VP40 selectively induces vesiculation from phosphatidylserine-enriched membranes.

Ebola virus is from the Filoviridae family of viruses and is one of the most virulent pathogens known with ∼ 60% clinical fatality. The Ebola virus negative sense RNA genome encodes seven proteins including viral matrix protein 40 (VP40), which is the most abundant protein found in the virions. Within infected cells VP40 localizes at the inner leaflet of the plasma membrane (PM), binds lipids, and regulates formation of new virus particles. Expression of VP40 in mammalian cells is sufficient to form virus-like particles that are nearly indistinguishable from the authentic virions. However, how VP40 interacts with the PM and forms virus-like particles is for the most part unknown. To investigate VP40 lipid specificity in a model of viral egress we employed giant unilamellar vesicles with different lipid compositions. The results demonstrate VP40 selectively induces vesiculation from membranes containing phosphatidylserine (PS) at concentrations of PS that are representative of the PM inner leaflet content. The formation of intraluminal vesicles was not significantly detected in the presence of other important PM lipids including cholesterol and polyvalent phosphoinositides, further demonstrating PS selectivity. Taken together, these studies suggest that PM phosphatidylserine may be an important component of Ebola virus budding and that VP40 may be able to mediate PM scission.

The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.

Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu(213), Ile(293), Leu(295), and Val(298) demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.

Effect of Combined HAART Plus ATT in Patients of Extrapulmonary Tuberculosis in HIV Patients and Effect on Quality of Life.

HIV is a disease which reduces human resistant to fight. When our immunity becomes so weak that we are not able to fight even the common commensals of our body, this leads to what is called as an opportunistic infection. Tuberculosis is one of those most common type of opportunistic infection and in this pulmonary tuberculosis is more common. Extrapulmonary tuberculosis with HIV is the area of lesser research. In this study we tried to prove the Prevalence of extrapulmonary tuberculosis in AIDS patients and also the effect of combined HAART and ATT on prevalence of extrapulmonary tuberculosis and also its effect on Quality of life of the patient. This is a retrospective study conducted in a tertiary centre in the Department of Otorhinolaryngology & head and neck surgery from January 2020 to December 2020. A total of 80 patients were studied for 1 year. Confirmed cases of AIDS with extra pulmonary tuberculosis in head and neck region were studied, keeping all ethical issues in mind and after complete treatment of ATT along with HAART. After treatment, a significant number of patients got resolved with EPTB and gave positive response on their quality of life. Not only pulmonary tuberculosis, extrapulmonary tuberculosis with HIV is on the rise nowadays. Combined treatment of both can help in decreasing prevalence of extrapulmonary tuberculosis and improves quality of life.

The Ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress.

Ebola virus, from the Filoviridae family has a high fatality rate in humans and nonhuman primates and to date, to the best of our knowledge, has no FDA approved vaccines or therapeutics. Viral protein 40 (VP40) is the major Ebola virus matrix protein that regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of VP40 membrane binding that are important for viral release remain to be elucidated. In this study, we used fluorescence quenching of a tryptophan on the membrane-binding interface with brominated lipids along with mutagenesis of VP40 to understand the depth of membrane penetration into lipid bilayers. Experimental results indicate that VP40 penetrates 8.1 Å into the hydrocarbon core of the plasma membrane bilayer. VP40 also induces substantial changes to membrane curvature as it tubulates liposomes and induces vesiculation into giant unilamellar vesicles, effects that are abrogated by hydrophobic mutations. This is a critical step in viral egress as cellular assays demonstrate that hydrophobic residues that penetrate deeply into the plasma membrane are essential for plasma membrane localization and virus-like particle formation and release from cells.

Analog and Digital Protocols for Functional Prosthetic Rehabilitation of Post-COVID-19 Rhino-Orbito Cerebral Mucormycosis Maxillofacial Defects: A Case Series.

The current study aims to present our clinical observations and experience gathered during the diagnosis, clinical presentation, medical/surgical treatment, and functional prosthetic rehabilitation of cases of rhino-orbital/cerebral Mucormycosis patients. Mucormycosis is an aggressive, life-threatening invasive fungal infection that occurs in people who are immune-compromised. The rise of ROCM during the second wave of the COVID-19 pandemic in India suggests a more effective association between Mucormycosis and the SARS-CoV-2 delta variant. The treatment strategy for Mucormycosis is an early diagnosis which is critical for a successful outcome. The initial step is to reduce or remove underlying risk factors followed by surgical excision and debridement of the afflicted tissues supplemented with antifungal medication. The first-line antifungal treatment is Liposomal Amphotericin B. Postsurgical resection defects are rehabilitated by either removable partial prosthesis (obturators) or by fixed zygomatic implant/patient-specific implant supported prosthesis. Management of ROCM requires a multidisciplinary approach. This case series highlights detailed medical, surgical, and functional prosthetic management modalities adopted by our team in managing such a dreaded disease which may be used as a tool for the formulation of standardized prevention and management/treatment/rehabilitation protocols in the future so that disease morbidity and mortality be reduced and an endemic outbreak could be averted.

Self-Assembly Properties of an Amphiphilic Phosphate Ester Prodrug Designed for the Treatment of COVID-19.

PF-07304814 is a water-soluble phosphate ester prodrug of a small molecule inhibitor for the SARS CoV-2 3CL protease designed for the treatment of COVID-19. The amphiphilicity and self-assembly behavior of the prodrug was investigated computationally and experimentally via multiple orthogonal techniques to better design formulations for intravenous infusion. The self-assembly of PF-07304814 into micellar structures enabled an increase in the solubility of lipophilic impurities by up to 1900x in clinically relevant formulations. The observed solubilization could help extend the drug product shelf-life and in use stability through inhibition of precipitation, without the need for solubilizing excipients. The work presented in this manuscript provides a roadmap for the characterization of prodrug self-assembly and highlights the potential for prodrug modifications to enhance solubility of both active ingredients and impurities and to extend drug product shelf-life.

Fine Governance of Difficult Tracheostomy in Difficult Airway with Stridor and Respiratory Distress.

Difficult airway is a commonly encountered problem in the anesthesia practice, might needing otolaryngologist expert in creation of surgical airway. Supraglottic airways, surgical or needle cricothyrotomy, high frequency jet ventilation, cardiopulmonary bypass (Tunstall in Can J Anaesth 36:611-613, 1989) are initial rescue measures in such scenario. But in otolaryngology practice, patient presenting with stridor having difficult airway and difficult tracheostomy concurrently will definitely pose problems resulting in life-threatening consequences. We report cases in which difficult airway and difficult tracheostomy coexisted. (1) upper tracheal stenosis following strangulation and intubation (2) short neck with obesity (3) blunt trauma to neck with surgical emphysema (4) deep neck space infection (5) Paediatric tracheostomy in faucial diphtheria. Though difficult surgical tracheostomy in difficult airway is challenging, the anticipation of complications and planning can minimise the difficulty in the technique.

Utility of Optical Forceps and Tracheotomy for Tracheobronchal Foreign Body.

Tracheobronchial foreign body is a life-threatening situation where quick decisions need to be taken. The preparedness for the same can never be over emphasized. This study was undertaken to understand the preference and utility of various types of foreign body forceps. This is a retrospective study in a tertiary care center. Data over 5 years (2016-2021) was analyzed to understand the utility of the optical forceps and the conventional (standard) forceps in the current scenario. The difficult cases where tracheotomy was done were also studied. Observations: 109 bronchoscopies were done during this period. In 90 patients optical forceps was used, flexible fiberoptic bronchoscope was used in 10 situations to locate and confirm the foreign body (FB) before extraction with standard or optical forceps. In 2 adult patients flexible fiber optic bronchoscope with forceps was used to extract, peripheral lying FB (pin). Standard (conventional) forceps with size 3 bronchoscope was used in 13 patients who were below 1 year. There were 2 postoperative mortalities. Tracheotomy was done in 6 patients, 4 were repaired primarily and in 2 tracheostomy tube was inserted. Conclusion: Optical forceps took care of 86% of foreign body. The Standard forceps have stood the test of time and are still useful. Having flexible fiberoptic in the armamentarium is a necessity now with newer challenges. Large swollen FB which cannot be extracted through glottis should be removed with tracheotomy rather than struggling at the glottis-sub glottis and have a complete obstruction. The optical forceps, standard forceps, ventilating rigid bronchoscope, flexible fiberoptic bronchoscope, Dormia basket, Fogarty's catheter are necessary in the armamentarium of an otolaryngologist and are not replaceable for one another.

Novel Use of Tracheostomy Shield for Emergency Tracheostomy in Covid 19 Era.

To evaluate the problems in doing emergency tracheostomy and tracheostomy care of patients with unknown COVID-19 status. Study the usefulness of the specially designed Tracheostomy Shield. A prospective hospital-based study was conducted at a tertiary care center in India treating COVID and Non COVID patients. The study was done from April 2020 to December 2020. A total of 80 tracheostomy were done using Tracheostomy shield. Open tracheostomies were 38 while 42 patients were already intubated (closed circuit). The Tracheostomy shield was also used in 380 tracheostomy tubes changes. Two patients were found COVID positive on RT-PCR after tracheostomy. There was no scope for testing all tube changes with RT-PCR. The use of our indigenously designed Tracheostomy shield has been effective in reducing the aerosol spread and there was no COVID transmission reported. The use of patient Tracheostomy shield is of uttermost importance in reducing the respiratory droplets splashing on to the surgeon and assistant while doing awake tracheostomy. It is extremely useful while changing tubes as there are chances of false negatives antigen test which might be miss leading. The use of Tracheostomy shield is very important in the current context of new and fast evolving respiratory infections.

Oleic- and docosahexaenoic acid-containing phosphatidylethanolamines differentially phase separate from sphingomyelin.

A central tenet of the lipid raft model is the existence of non-raft domains. In support of this view, we have established in model membranes that a phosphatidylethanolamine (PE)-containing docosahexaenoic acid (DHA) forms organizationally distinct non-raft domains in the presence of sphingomyelin (SM) and cholesterol (Chol). We have shown that formation of DHA-rich domains is driven by unfavorable molecular interactions between the rigid Chol molecule and the highly flexible DHA acyl chain. However, the molecular interactions between SM and the DHA-containing PE, which could also contribute to the formation of DHA-rich non-raft domains, have not been sufficiently investigated. To address this issue, we use differential scanning calorimetry (DSC) to study the phase behavior of mixtures of SM with either 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE), an oleic acid (OA)-containing control, over a wide range of concentrations. Deconvolution of binary DSC scans shows that both 16:0-22:6PE and 16:0-18:1PE phase separate from SM. Analysis of transition temperatures and partial phase diagrams, constructed from the DSC scans for the first time, shows that 16:0-22:6PE displays greater non-ideal mixing with SM compared to 16:0-18:1PE. Our findings support a model in which DHA- and OA-containing PEs differentially phase separate from SM over a wide range of molar ratios to initiate the formation of non-raft domains, which is greatly enhanced by DHA, but not OA, in the presence of cholesterol.

Effect of trans unsaturation on molecular organization in a phospholipid membrane.

We employed solid state (2)H NMR, complemented by computer simulations, to compare molecular organization in model membranes composed of 1-elaidoyl-2-stearoylphosphatidylcholine (t18:1-18:0PC), 1-oleoyl-2-stearoylphosphatidylcholine (c18:1-18:0PC), and 1,2-distearoylphosphatidylcholine (18:0-18:0PC). These phospholipids have elaidic acid (EA) containing a trans double bond, oleic acid (OA) containing a cis double bond, and saturated stearic acid (SA), respectively, at the sn-1 position and were synthesized with perdeuterated SA at the sn-2 position. The temperature of the chain melting transition is depressed less for t18:1-18:0PC (31.5 degrees C) than c18:1-18:0PC (7 degrees C) relative to 18:0-18:0PC (53 degrees C), reflecting the smaller deviation from the linear conformation produced by a trans as opposed to cis double bond. Acyl chain order in t18:1-18:0PC (S(CD) = 0.135) in the liquid crystalline state is much closer to that of c18:1-18:0PC (S(CD) = 0.128) than that of the substantially more ordered 18:0-18:0PC (S(CD) > 0.156), which is attributed to the reduced energy barrier to rotation about the C-C single bonds next to either a trans or cis carbon double bond. A conformation that somewhat resembles a saturated chain and an intrinsic disorder approaching that of a cis unsaturated chain characterize EA and, we speculate, may play a role in the adverse impact dietary trans fatty acids (TFA) have on biological function.

A Study of Toluidine Blue Staining in Suspected Oral Malignancies in Patients Presenting to Tertiary Care Hospital in Central India.

Oral malignancies are a major cause of morbidity and mortality in worldwide and especially in Indian subcontinent. India stands at first position in total number of oral cancers in worldwide. Hence it is the need of hour to have safe and reliable screening test for early diagnosis of oral malignancies. Toluidine blue (TB) staining is an ideal screening test with high sensitivity and specificity. This study carried out in our department with objective to study efficacy of TB in oral malignancies and potential oral malignant lesions and also to evaluate and compare TB staining with histopathological examination of lesion. This prospective study conducted on 183 patients who attended ENT department. Out of 183 patients 146 (79.8%) were positive with TB and 37 (20.2%) were not stained with TB. Out of 146 positive patients 134 were turned out to be positive for malignancy in histopathological examination. Out of 37 negative patients 5 were positive for malignancy in histopathological examination and 32 were negative. Out of 183 patients 139 (76%) were positive whereas 44 (24%) were found negative for malignancy. In present study clinical examination of oral cavity reveal most of the patients had leukoplakis (37.7%) followed by swelling (26.8%), ulcer (26.2%). Positive and negative predictive value of TB was 91.8% and 86.5% respectively whereas positive and negative predictive value of histopathology was 96.4% and 72.7% respectively. TB stain is cheap, quick and easily available readymade kit and no side effects and so have excellent patient compliance so it can be best utilized as a premier tool in initial detection of suspected oral malignancy.

Docosahexaenoic acid enhances segregation of lipids between : 2H-NMR study.

Solid-state (2)H-NMR of [(2)H(31)]-N-palmitoylsphingomyelin ([(2)H(31)]16:0SM, PSM*), supplemented by differential scanning calorimetry, was used for the first time, to our knowledge, to investigate the molecular organization of the sphingolipid in 1:1:1 mol mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE, POPE) or 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE, PDPE) and cholesterol. When compared with (2)H-NMR data for analogous mixtures of [(2)H(31)]16:0-18:1PE (POPE*) or [(2)H(31)]16:0-22:6PE (PDPE*) with egg SM and cholesterol, molecular interactions of oleic acid (OA) versus docosahexaenoic acid (DHA) are distinguished, and details of membrane architecture emerge. SM-rich, characterized by higher-order, and PE-rich, characterized by lower-order, domains <20 nm in size are formed in the absence and presence of cholesterol in both OA- and DHA-containing membranes. Although acyl chain order within both domains increases on the addition of sterol to the two systems, the resultant differential in order between SM- and PE-rich domains is almost a factor of 3 greater with DHA than with OA. Our interpretation is that the aversion that cholesterol has for DHA--but not for OA--excludes the sterol from DHA-containing, PE-rich (nonraft) domains and excludes DHA from SM-rich/cholesterol-rich (raft) domains. We attribute, in part, the diverse health benefits associated with dietary consumption of DHA to an alteration in membrane domains.

A Study of Clinicopathological Profile of Patients of Hoarseness of Voice Presenting to Tertiary Care hospital.

Hoarseness is subjective term used to describe abnormal voice quality which may occur due to many causes because it is just a symptom. Proper knowledge and clinicopathological profile is important to treat the underlying pathology. This prospective study was carried out in 100 patients presented in Department of ENT, Gandhi Medical College, Bhopal from April 2013 to September 2014 with complaint of hoarseness of voice for more than 15 days. Objective of this study is to study incidence, duration and sex predilection for hoarseness of voice. Also to study different etiological and predisposing factors for hoarseness of voice. After taking detailed history of the patient, complete examination of ear, nose and throat has been carried out. Indirect laryngoscopy, direct laryngoscopy, FOFE is done. Any suspicious tissue is sent for histopathological evaluation. X-ray soft tissue neck and if required CT SCAN larynx is done. Out of 100 patients maximum were from 50 to 70 years age group. 89% were males while only 11% were females. Labourers (37%) and farmers (32%) were the major groups affected. Smoking habit found in 60% of patients and tobacco chewing in 33% of patients and both, also having. Most common cause for hoarseness was found out to be laryngeal neoplasms of which supraglottic growth being commonest (37%) in our study. Hoarseness of voice may be present due to various underlying pathologies. So proper diagnosis, through detailed history, clinical examination and investigations is warranted to find out the cause and starting treatment.

Effect of Nasal Allergy Over Spirometric Parameters: A Prospective Study.

Nasal allergy is characterized by an IgE mediated inflammatory response of nasal mucosa to allergens and it has a close association with Asthma. Nasal allergy has been demonstrated to be a strong risk factor for the onset of asthma in adults. Spirometric parameters like Forced expiratory volume at timed interval of 1 s (FEV1) and forced expiratory flow (FEF25-75 %) are impaired in patients with nasal allergy or allergic rhinitis. The FEF25-75 % has been evidenced to be a reliable marker of early bronchial impairment in nasal allergy. Nasal allergy may be considered as the first step of the progression of respiratory allergy towards asthma. It has been demonstrated that FEF25-75 % is useful in predicting the presence of airway hyper responsiveness.It may be a more sensitive indicator of chronic airway obstruction than FEV1 and is considered as a risk factor for the persistence of respiratory symptoms in asthmatic patients. The impact of allergic rhinitis or nasal allergy on asthma (ARIA) guidelines, clearly underlined the role of allergic rhinitis as risk factor for asthma development. The possible presence of spirometric abnormalities in patient with allergic rhinitis has been well documented. So keeping this in mind, present study is undertaken to evaluate the impairment of spirometric parameters, like FEV1, FEF25-75 %, and forced vital capacity, in patients with nasal allergy and to predict the presence of airway hyper responsiveness.

A loop region in the N-terminal domain of Ebola virus VP40 is important in viral assembly, budding, and egress.

Ebola virus (EBOV) causes viral hemorrhagic fever in humans and can have clinical fatality rates of ~60%. The EBOV genome consists of negative sense RNA that encodes seven proteins including viral protein 40 (VP40). VP40 is the major Ebola virus matrix protein and regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane of human cells to regulate viral budding where VP40 can produce virus like particles (VLPs) without other Ebola virus proteins present. The mechanistic details, however, of VP40 lipid-interactions and protein-protein interactions that are important for viral release remain to be elucidated. Here, we mutated a loop region in the N-terminal domain of VP40 (Lys127, Thr129, and Asn130) and find that mutations (K127A, T129A, and N130A) in this loop region reduce plasma membrane localization of VP40. Additionally, using total internal reflection fluorescence microscopy and number and brightness analysis we demonstrate these mutations greatly reduce VP40 oligomerization. Lastly, VLP assays demonstrate these mutations significantly reduce VLP release from cells. Taken together, these studies identify an important loop region in VP40 that may be essential to viral egress.

Molecular organization of cholesterol in unsaturated phosphatidylethanolamines: X-ray diffraction and solid state 2H NMR reveal differences with phosphatidylcholines.

The major mammalian plasma membrane lipids are phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and cholesterol. Whereas PC-cholesterol interactions are well studied, far less is known about those between PE and cholesterol. Here, we investigated the molecular organization of cholesterol in PEs that vary in their degree of acyl chain unsaturation. For heteroacid sn-1 saturated (palmitoyl), sn-2 unsaturated (various acyl chain) PEs, cholesterol solubility determined by X-ray diffraction was essentially identical with 1 (oleoyl, 51 +/- 3 mol %) and 2 (linoleoyl, 49 +/- 2 mol %) double bonds before decreasing progressively with 4 (arachidonyl, 41 +/- 3 mol %) and 6 (docosahexaenoyl, 31 +/- 3 mol %) double bonds. With 6 double bonds in each chain, cholesterol solubility was further reduced to 8.5 +/- 1 mol %. However, (2)H NMR experiments established that the orientation of cholesterol in the same heteroacid PE membranes was unaffected by the degree of acyl chain unsaturation. A tilt angle of 15 +/- 1 degrees was measured when equimolar [3alpha-(2)H(1)]cholesterol was added, regardless of the number of double bonds in the sn-2 chain. The finding that solubility of cholesterol in sn-1 saturated PEs depends on the amount of polyunsaturation in the sn-2 chain of PE differs from the equivalent PCs that universally incorporate approximately 50 mol % sterol. Unlike PCs, a differential in affinity for cholesterol and tendency to drive lateral segregation is inferred between polyunsaturated PEs. This distinction may have biological implications reflected by the health benefits of dietary polyunsaturated fatty acids that are often taken up into PE > PC.