Environmental SARS-CoV-2 contamination in hospital rooms of patients with acute COVID-19.

OBJECTIVE: Data on the transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remain conflicting. Airborne transmission is still debated. However, hospital risk control requires better understanding of the different modes of transmission. This study aimed to evaluate the frequency of, and factors associated with, environmental air and surface contamination in the rooms of patients with coronavirus disease 2019 in the acute phase of the disease. METHODS: Sixty-five consecutive patients were included in this study. For each patient, seven room surfaces, air 1 m and 3 m from the patient's head, the inner surface of the patient's mask, and the outer surface of healthcare workers' (HCW) masks were sampled. Environmental contamination was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for SARS-CoV-2 RNA on surfaces, air and masks. A viral isolation test was performed on Vero cells for samples with an RT-qPCR cycle threshold (Ct) ≤37. RESULTS: SARS-CoV-2 RNA was detected by RT-qPCR in 34%, 12%, 50% and 10% of surface, air, patient mask and HCW mask samples, respectively. Infectious virus was isolated in culture from two samples among the 85 positive samples with Ct ≤37. On multi-variate analysis, only a positive result for SARS-CoV-2 RT-qPCR for patients' face masks was found to be significantly associated with surface contamination (odds ratio 5.79, 95% confidence interval 1.31-25.67; P=0.025). CONCLUSION: This study found that surface contamination by SARS-CoV-2 was more common than air and mask contamination. However, viable virus was rare. The inner surface of a patient's mask could be used as a marker to identify those at higher risk of contamination.

Effect of the HIV-1 fusion peptide on the mechanical properties and leaflet coupling of lipid bilayers.

The fusion peptide (FP) of the human immunodeficiency virus (HIV) is part of the N-terminus of the viral envelope glycoprotein gp41 and is believed to play an important role in the viral entry process. To understand the immediate effect of this peptide on the cell membrane, we have studied the influence of the synthetic FP sequence FP23 on the mechanical properties of model lipid bilayers. For this purpose, giant unilamellar vesicles were prepared from the unsaturated lipid dioleoylphosphatidylcholine mixed in various molar ratios with FP23. The bending stiffness of the vesicles was measured with two different methods: fluctuation analysis and aspiration with micropipettes. The data obtained from both of these approaches show that the bending stiffness of the membrane decreases gradually with increasing concentration of the FP23 in the bilayer. Low concentrations of only a few mol% FP23 are sufficient to decrease the bending stiffness of the lipid bilayer by about a factor of 2. Finally, data obtained for the stretching elasticity modulus of the membrane suggest that the peptide insertion decreases the coupling between the two leaflets of the bilayer.

Plasmids encoding the mucosal chemokines CCL27 and CCL28 are effective adjuvants in eliciting antigen-specific immunity in vivo.

A hurdle facing DNA vaccine development is the ability to generate strong immune responses systemically and at local immune sites. We report a novel systemically administered DNA vaccination strategy using intramuscular codelivery of CCL27 or CCL28, which elicited elevated peripheral IFN-gamma and antigen-specific IgG while driving antigen-specific T-cell secretion of cytokine and antibody production in the gut-associated lymphoid tissue and lung. This strategy resulted in induction of long-lived antibody responses that neutralized influenza A/PR8/34 and protected mice from morbidity and mortality associated with a lethal intranasal viral challenge. This is the first example of the use of CCL27 and CCL28 chemokines as adjuvants to influence a DNA vaccine strategy, suggesting further examination of this approach for manipulation of vaccine-induced immunity impacting both quality and phenotype of responses.

Comparative study of outcome of type I tympanoplasty in dry and wet ear.

Chronic suppurative otitis media is a common condition seen in patients attending the otolaryngology clinic. The discharging ear presents the otologist with the dilemma of operating on it or not. This due to the widespread belief that the success rate while doing tympanoplasty on wet ears is decidedly inferior. To evaluate this fact we conducted a study to compare outcomes of type 1 tympanoplasty in dry and wet ears. Wet ear meant that the patient had a mild mucoid discharge which was negative on culture. Type 1 tympanoplasty was done in all patients under local anesthesia using temporalis fascia graft and by underlay technique. We conclude that the presence of discharge in the ear at the time of operation does not interfere with the results of tympanoplasty, but it should be mucoid and scanty.

Tumoral calcinosis--clinicopathological study of five cases.

Five cases of tumoral calcinosis were studied during the period of January-february 1999 in MIMER medical college, Talegaon, a rural place 35kms. from Pune. All the Five patients were females residing in nonendemic area for Dracunculosis. They were from 5th-6th decade. They were otherwise healthy and had normal serum calcium and phosphorus levels and no eosinophilia. All had large, hard subcutaneous lump around hip joint. The skin overlying the swelling was normal. Histologically all cases showed similar morphology, the lesions were composed of large and small deposits of calcium. The foreign-body giant cell reaction was seen in two cases. There were no eosinophils and lymphocytes. On multiple sectioning none of the cases revealed any evidence of dead or living parasite. Old and recent necrosis was absent. These cases are presented since the condition is comparatively rare and it appeared in crops in our Institute.

Reconstruction of mandible with free fibular vascular graft.

This is a case report of ameloblastoma of left mandible. It was treated with left extended hemimandibulectomy with free fibular vascular graft.

Creating a library of generalized Fourier sampling patterns for irregular 2D regions of support.

Multiple-region MRI (mrMRI) represents a generalization of the Shannon sampling theorem to permit sparse k-space sampling whenever the scanned object or its high-contrast edges are confined to multiple known regions. Use of an optimal mrMRI sampling pattern produces an image with root-mean-squared (RMS) noise over the supporting regions equal to the RMS noise in a conventional Fourier image with the same total area of support. Analytical solutions for such sampling patterns have been described previously for all arrangements of two or three (noncollinear) supporting regions. This work describes a robust numerical method for creating a library of optimal and near-optimal mrMRI sampling patterns for more complicated geometries. The average noise amplification over all sampling patterns in the demonstration library was only 4%, with 30% of the sampling patterns resulting in no noise amplification whatsoever.

The thermotropic phase behavior of cationic lipids: calorimetric, infrared spectroscopic and X-ray diffraction studies of lipid bilayer membranes composed of 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP).

The thermotropic phase behavior of lipid bilayer model membranes composed of the cationic lipid 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP) was examined by differential scanning calorimetry, infrared spectroscopy and X-ray diffraction. Aqueous dispersions of this lipid exhibit a highly energetic endothermic transition at 38.4 degrees C upon heating and two exothermic transitions between 20 and 30 degrees C upon cooling. These transitions are accompanied by enthalpy changes that are considerably greater than normally observed with typical gel/liquid--crystalline phase transitions and have been assigned to interconversions between lamellar crystalline and lamellar liquid--crystalline forms of this lipid. Both infrared spectroscopy and X-ray diffraction indicate that the lamellar crystalline phase is a highly ordered, substantially dehydrated structure in which the hydrocarbon chains are essentially immobilized in a distorted orthorhombic subcell. Upon heating to temperatures near 38.4 degrees C, this structure converts to a liquid-crystalline phase in which there is excessive swelling of the aqueous interlamellar spaces owing to charge repulsion between, and undulations of, the positively charged lipid surfaces. The polar/apolar interfaces of liquid--crystalline DM-TAP bilayers are not as well hydrated as those formed by other classes of phospho- and glycolipids. Such differences are attributed to the relatively small size of the polar headgroup and its limited capacity for interaction with moieties in the bilayer polar/apolar interface.

Method for obtaining structure and interactions from oriented lipid bilayers.

Precise calculations are made of the scattering intensity I(q) from an oriented stack of lipid bilayers using a realistic model of fluctuations. The quantities of interest include the bilayer bending modulus Kc, the interbilayer interaction modulus B, and bilayer structure through the form factor F(qz). It is shown how Kc and B may be obtained from data at large q(z) where fluctuations dominate. Good estimates of F(qz) can be made over wide ranges of q(z) by using I(q) in q regions away from the peaks and for q(r) not equal0 where details of the scattering domains play little role. Rough estimates of domain sizes can also be made from smaller q(z) data. Results are presented for data taken on fully hydrated, oriented DOPC bilayers in the L(alpha) phase. These results illustrate the advantages of oriented samples compared to powder samples.

Structure of lipid bilayers.

The quantitative experimental uncertainty in the structure of fully hydrated, biologically relevant, fluid (L(alpha)) phase lipid bilayers has been too large to provide a firm base for applications or for comparison with simulations. Many structural methods are reviewed including modern liquid crystallography of lipid bilayers that deals with the fully developed undulation fluctuations that occur in the L(alpha) phase. These fluctuations degrade the higher order diffraction data in a way that, if unrecognized, leads to erroneous conclusions regarding bilayer structure. Diffraction measurements at high instrumental resolution provide a measure of these fluctuations. In addition to providing better structural determination, this opens a new window on interactions between bilayers, so the experimental determination of interbilayer interaction parameters is reviewed briefly. We introduce a new structural correction based on fluctuations that has not been included in any previous studies. Updated measurements, such as for the area compressibility modulus, are used to provide adjustments to many of the literature values of structural quantities. Since the gel (L(beta)') phase is valuable as a stepping stone for obtaining fluid phase results, a brief review is given of the lower temperature phases. The uncertainty in structural results for lipid bilayers is being reduced and best current values are provided for bilayers of five lipids.

Clarification of the ripple phase of lecithin bilayers using fully hydrated, aligned samples.

Aligned samples of lipid bilayers have been fully hydrated from water vapor in a different type of x-ray chamber. Our use of aligned samples resolves issues concerning the ripple phase that were ambiguous from previous powder studies. In particular, our x-ray diffraction data conclusively demonstrate that, on cooling from the L alpha to the P beta' phase, both chiral and racemic samples of dipalmitoyl phosphatidylcholine (DPPC) exhibit phase coexistence of long and short ripples with a ripple wavelength ratio lambda L/lambda S approximately 1.8. Moreover, the long ripple always forms an orthorhombic unit cell (gamma L = 90 degrees), strongly supporting the possibility that these ripples are symmetric. In contrast, gamma S for short ripples was consistently different from 90 degrees, implying asymmetric ripples. We continue to find no evidence that chirality affects the structure of rippled bilayers. The relative thermodynamic stability of the two types of ripples was investigated and a qualitative free energy diagram is given in which the long ripple phase is metastable. Finally, we suggest a kinetic mechanism, involving loss of water, that promotes formation of the metastable long ripple phase for special thermal protocols.

Lipid bilayer structure.

Fluctuations, inherent in flexible and biologically relevant lipid bilayers, make quantitative structure determination challenging. Shortcomings in older methods of structure determination have been realized and new methodologies have been introduced that take fluctuations into account. The large uncertainty in literature values of lipid bilayer structural parameters is being reduced.

Re-analysis of magic angle spinning nuclear magnetic resonance determination of interlamellar waters in lipid bilayer dispersions.

A recent method to obtain the number of water molecules of hydration of multilamellar lipid vesicles using magic angle spinning nuclear magnetic resonance has been re-examined. The previous interpretation divided the water into bulk and interlamellar water and ignored water in defects (lakes) that are intrinsic to multilamellar lipid vesicles; the result was inconsistent with x-ray results for the lipid DOPC. The new interpretation takes advantage of the reduction of lake water with increased spinning and it uses osmotic pressure measurements to determine the loss of interlamellar water. The new result for DOPC from magic angle spinning is consistent with x-ray results.

Locally focused contrast-enhanced carotid MRA.

With conventional Fourier transform (FT) magnetic resonance imaging (MRI), it is difficult to perform contrast-enhanced three-dimensional (3D) MR angiography (MRA) with the temporal and spatial resolution necessary to depict the carotid arteries. However, locally focused (LF) MRI is a more efficient method that utilizes prior knowledge of the image content to reconstruct images from sparse k-space samples. In this paper, we show how LF MRI can be used to perform high-resolution gadolinium (Gd)-enhanced 3D carotid MRA in less than 10 seconds. First, the accuracy of the technique was demonstrated by comparing LF and conventional (FT) images of a vascular phantom. Then the method was used to perform Gd-enhanced 3D MRA of a patient's carotid arteries. Instead of using bolus timing, the arterial phase was retrospectively identified in a consecutive series of images, just as in X-ray angiography.

Multiple region MRI.

Traditional Fourier MR imaging (FT MRI) utilizes the Whittaker-Kotel'nikov-Shannon (WKS) sampling theorem. This theorem specifies the spatial frequency components which need to be measured to reconstruct an image with a known field of view (FOV). In this paper, we generalize this result in order to find the optimal k-space sampling for images that vanish except in multiple, possibly non-adjacent regions within the FOV. This provides the basis for "multiple region MRI" (mrMRI), a method of producing such images from a traction of the k-space samples required by the WKS theorem. Image reconstruction does not suffer from noise amplification and can be performed rapidly with fast Fourier transforms, just as in conventional FT MRI. The mrMRI method can also be used to reconstruct images that have low spatial-frequency components throughout the entire FOV and high spatial frequencies (i.e. edges) confined to multiple small regions. The greater efficiency of mrMRI sampling can be parlayed into increased temporal or spatial resolution whenever the imaged objects have signal or "edge" intensity confined to multiple small portions of the FOV. Possible areas of application include MR angiography (MRA), interventional MRI, functional MRI, and spectroscopic MRI. The technique is demonstrated by using it to acquire Gd-enhanced first-pass 3D MRA images of the carotid arteries without the use of bolus-timing techniques.

Polymorphism in myristoylpalmitoylphosphatidylcholine.

This study focuses on the mixed-chain lipid myristoylpalmitoylphosphatidylcholine (MPPC) near full hydration. The lipid, synthesized according to the procedure of (Mason et al., 1981a, has a low degree of acyl chain migration. When MPPC is temperature-jumped (T-jumped) from the L alpha phase (T = 38 degrees C) to T = 20 degrees C or below, a subgel phase forms; this formation takes less than 1 h at a temperature below T = 12 degrees C. The subgel remains stable up to T = 29 degrees C. When MPPC is T-jumped from the L alpha phase to T = 24 degrees C or above, a ripple phase forms with coexisting ripple wavelengths of 240 A and 130 A. In contrast, when MPPC is melted from the subgel phase, the ripple phase is characterized by bilayers having a single ripple wavelength of 130 A. In agreement with earlier studies (Stumpel et al., 1983; Serrallach et al., 1984. Structure and thermotropic properties of mixed-chain phosphatidylcholine bilayer membranes. Biochemistry 23:713-720.), no stable gel phase was observed. Instead, an ill-defined low-angle X-ray pattern is initially observed, which gradually transforms into the subgel phase below 20 degrees C, or into the ripple phase above 24 degrees C. In the wide-angle X-ray diffraction, a single peak is observed, similar to the ripple phase wide-angle pattern, that either persists above 24 degrees C or transforms into a multi-peaked subgel wide-angle pattern below 20 degrees C. The absence of a gel phase can be understood phenomenologically as the relative dominance of the subgel phase in mixed-chain PCs compared to same-chain PCs. The subgel structure and molecular interactions responsible for this comparative behavior are interesting open issues.

Locally focused MRI of interventions.

Certain interventional MR procedures would benefit from T2-weighted imaging because of the sensitivity of T2-weighted images to tissue damage and target lesion contrast. To acquire such images with reasonable temporal resolution, a single-shot acquisition should be used because of the inherently long TR needed for T2 weighting. Unfortunately, most scanners require long readout times (eg, greater than 150 msec) and high bandwidths (eg, greater than 120 kHz) to perform conventional single-shot imaging with high spatial resolution. The resulting images are thus degraded by unacceptable artifacts and noise levels. This study illustrates how to create locally focused MR images that have high spatial resolution in a region of interest and lower spatial resolution elsewhere in the image. Because these images can be created from sparse k-space data, a scanner with modest gradients (eg, 10 mT/m maximal amplitude, 500 microsec minimal rise time) can acquire them after a single excitation with relatively short readout time and low bandwidth. This technique may make it practical to monitor interventions with T2-weighted imaging. The method was illustrated by reconstructing dynamic changes, which were simulated experimentally by moving objects in the vicinity of a normal human head.

Fluid phase structure of EPC and DMPC bilayers.

X-ray diffraction data taken at high instrumental resolution were obtained for EPC and DMPC under various osmotic pressures, primarily at T = 30 degrees C. The headgroup thickness DHH was obtained from relative electron density profiles. By using volumetric results and by comparing to gel phase DPPC we obtain areas AEPCF = 69.4 +/- 1.1 A2 and ADMPCF = 59.7 +/- 0.2 A2. The analysis also gives estimates for the areal compressibility KA. The AF results lead to other structural results regarding membrane thickness and associated waters. Using the recently determined absolute electrons density profile of DPPC, the AF results also lead to absolute electron density profiles and absolute continuous transforms magnitude of F(q) for EPC and DMPC. Limited measurements of temperature dependence show directly that fluctuations increase with increasing temperature and that a small decrease in bending modulus KC accounts for the increased water spacing reported by Simon et al. (1995) Biophys. J. 69, 1473-1483.

Structure and interactions of fully hydrated dioleoylphosphatidylcholine bilayers.

This study focuses on dioleoylphosphatidylcholine (DOPC) bilayers near full hydration. Volumetric data and high-resolution synchrotron x-ray data are used in a method that compares DOPC with well determined gel phase dipalmitoylphosphatidylcholine (DPPC). The key structural quantity obtained is fully hydrated area/lipid A0 = 72.2 +/- 1.1 A2 at 30 degrees C, from which other quantities such as thickness of the bilayer are obtained. Data for samples over osmotic pressures from 0 to 56 atmospheres give an estimate for the area compressibility of KA = 188 dyn/cm. Obtaining the continuous scattering transform and electron density profiles requires correction for liquid crystal fluctuations. Quantitation of these fluctuations opens an experimental window on the fluctuation pressure, the primary repulsive interaction near full hydration. The fluctuation pressure decays exponentially with water spacing, in agreement with analytical results for soft confinement. However, the ratio of decay length lambda(fl) = 5.8 A to hydration pressure decay length lambda = 2.2 A is significantly larger than the value of 2 predicted by analytical theory and close to the ratio obtained in recent simulations. We also obtain the traditional osmotic pressure versus water spacing data. Our analysis of these data shows that estimates of the Hamaker parameter H and the bending modulus Kc are strongly coupled.

DMSO produces a new subgel phase in DPPC: DSC and X-ray diffraction study.

Equilibrium phases and the kinetics of subgel phase transformation of dipalmitoylphosphatidylcholine (DPPC) hydrated with mixtures of dimethylsulfoxide (DMSO)/water have been studied using differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The rate of gel-to-subgel transformation is decreased with a small increase in X, the DMSO/water mole fraction, but then speeds up and becomes faster than in pure water by X = 0.16. The DSC scans show multiple subgel peaks, some of which can be attributed to impacted domain growth. For X greater than 0.10, XRD shows that there is a new, stable subgel phase, S, which also accounts for some of the multiplicity of DSC peaks. Our electron density profiles show that the thickness of the bilayer in the S phase is greater than in the usual C subgel phase. We suggest that the S subgel phase is characterized by different headgroup ordering and smaller chain tilt angle than in the C subgel phase. Electron density profiles show that increasing X decreases the water space between bilayers in all phases, subgel, gel and fluid (L alpha). For X = 0.20, a different gel phase is also observed that may be due to subtle changes in the orientation of chain tilt first observed in partially dehydrated DMPC. The dehydrating effect of DMSO explains the results of a previous study, confirmed in this study, that increasing the concentration of DMSO raises the main transition temperature and eliminates the ripple phase.