Cryo-EM images of phase-separated lipid bilayer vesicles analyzed with a machine-learning approach.

Lateral lipid heterogeneity (i.e., raft formation) in biomembranes plays a functional role in living cells. Three-component mixtures of low- and high-melting lipids plus cholesterol offer a simplified experimental model for raft domains in which a liquid-disordered (Ld) phase coexists with a liquid-ordered (Lo) phase. Using such models, we recently showed that cryogenic electron microscopy (cryo-EM) can detect phase separation in lipid vesicles based on differences in bilayer thickness. However, the considerable noise within cryo-EM data poses a significant challenge for accurately determining the membrane phase state at high spatial resolution. To this end, we have developed an image-processing pipeline that utilizes machine learning (ML) to predict the bilayer phase in projection images of lipid vesicles. Importantly, the ML method exploits differences in both the thickness and molecular density of Lo compared to Ld, which leads to improved phase identification. To assess accuracy, we used artificial images of phase-separated lipid vesicles generated from all-atom molecular dynamics simulations of Lo and Ld phases. Synthetic ground-truth data sets mimicking a series of compositions along a tieline of Ld + Lo coexistence were created and then analyzed with various ML models. For all tieline compositions, we find that the ML approach can correctly identify the bilayer phase with >90% accuracy, thus providing a means to isolate the intensity profiles of coexisting Ld and Lo phases, as well as accurately determine domain-size distributions, number of domains, and phase-area fractions. The method described here provides a framework for characterizing nanoscopic lateral heterogeneities in membranes and paves the way for a more detailed understanding of raft properties in biological contexts.

Visualizing lipid membrane structure with cryo-EM: past, present, and future.

The development of electron cryomicroscopy (cryo-EM) has evolved immensely in the last several decades and is now well-established in the analysis of protein structure both in isolation and in their cellular context. This review focuses on the history and application of cryo-EM to the analysis of membrane architecture. Parallels between the levels of organization of protein structure are useful in organizing the discussion of the unique parameters that influence membrane structure and function. Importantly, the timescales of lipid motion in bilayers with respect to the timescales of sample vitrification is discussed and reveals what types of membrane structure can be reliably extracted in cryo-EM images of vitrified samples. Appreciating these limitations, a review of the application of cryo-EM to examine the lateral organization of ordered and disordered domains in reconstituted and biologically derived membranes is provided. Finally, a brief outlook for further development and application of cryo-EM to the analysis of membrane architecture is provided.

Multi-contrast high-field quality image synthesis for portable low-field MRI using generative adversarial networks and paired data.

Introduction: Portable low-field strength (64mT) MRI scanners promise to increase access to neuroimaging for clinical and research purposes, however these devices produce lower quality images compared to high-field scanners. In this study, we developed and evaluated a deep learning architecture to generate high-field quality brain images from low-field inputs using a paired dataset of multiple sclerosis (MS) patients scanned at 64mT and 3T. Methods: A total of 49 MS patients were scanned on portable 64mT and standard 3T scanners at Penn (n=25) or the National Institutes of Health (NIH, n=24) with T1-weighted, T2-weighted and FLAIR acquisitions. Using this paired data, we developed a generative adversarial network (GAN) architecture for low- to high-field image translation (LowGAN). We then evaluated synthesized images with respect to image quality, brain morphometry, and white matter lesions. Results: Synthetic high-field images demonstrated visually superior quality compared to low-field inputs and significantly higher normalized cross-correlation (NCC) to actual high-field images for T1 (p=0.001) and FLAIR (p<0.001) contrasts. LowGAN generally outperformed the current state-of-the-art for low-field volumetrics. For example, thalamic, lateral ventricle, and total cortical volumes in LowGAN outputs did not differ significantly from 3T measurements. Synthetic outputs preserved MS lesions and captured a known inverse relationship between total lesion volume and thalamic volume. Conclusions: LowGAN generates synthetic high-field images with comparable visual and quantitative quality to actual high-field scans. Enhancing portable MRI image quality could add value and boost clinician confidence, enabling wider adoption of this technology.

Deconvoluting complex correlates of COVID-19 severity with a multi-omic pandemic tracking strategy.

The SARS-CoV-2 pandemic has differentially impacted populations across race and ethnicity. A multi-omic approach represents a powerful tool to examine risk across multi-ancestry genomes. We leverage a pandemic tracking strategy in which we sequence viral and host genomes and transcriptomes from nasopharyngeal swabs of 1049 individuals (736 SARS-CoV-2 positive and 313 SARS-CoV-2 negative) and integrate them with digital phenotypes from electronic health records from a diverse catchment area in Northern California. Genome-wide association disaggregated by admixture mapping reveals novel COVID-19-severity-associated regions containing previously reported markers of neurologic, pulmonary and viral disease susceptibility. Phylodynamic tracking of consensus viral genomes reveals no association with disease severity or inferred ancestry. Summary data from multiomic investigation reveals metagenomic and HLA associations with severe COVID-19. The wealth of data available from residual nasopharyngeal swabs in combination with clinical data abstracted automatically at scale highlights a powerful strategy for pandemic tracking, and reveals distinct epidemiologic, genetic, and biological associations for those at the highest risk.

Sensitivity of portable low-field magnetic resonance imaging for multiple sclerosis lesions.

Magnetic resonance imaging (MRI) is a fundamental tool in the diagnosis and management of neurological diseases such as multiple sclerosis (MS). New portable, low-field strength, MRI scanners could potentially lower financial and technical barriers to neuroimaging and reach underserved or disabled populations, but the sensitivity of these devices for MS lesions is unknown. We sought to determine if white matter lesions can be detected on a portable 64mT scanner, compare automated lesion segmentations and total lesion volume between paired 3T and 64mT scans, identify features that contribute to lesion detection accuracy, and explore super-resolution imaging at low-field. In this prospective, cross-sectional study, same-day brain MRI (FLAIR, T1w, and T2w) scans were collected from 36 adults (32 women; mean age, 50 ± 14 years) with known or suspected MS using Siemens 3T (FLAIR: 1 mm isotropic, T1w: 1 mm isotropic, and T2w: 0.34-0.5 × 0.34-0.5 × 3-5 mm) and Hyperfine 64mT (FLAIR: 1.6 × 1.6 × 5 mm, T1w: 1.5 × 1.5 × 5 mm, and T2w: 1.5 × 1.5 × 5 mm) scanners at two centers. Images were reviewed by neuroradiologists. MS lesions were measured manually and segmented using an automated algorithm. Statistical analyses assessed accuracy and variability of segmentations across scanners and systematic scanner biases in automated volumetric measurements. Lesions were identified on 64mT scans in 94% (31/33) of patients with confirmed MS. The average smallest lesions manually detected were 5.7 ± 1.3 mm in maximum diameter at 64mT vs 2.1 ± 0.6 mm at 3T, approaching the spatial resolution of the respective scanner sequences (3T: 1 mm, 64mT: 5 mm slice thickness). Automated lesion volume estimates were highly correlated between 3T and 64mT scans (r = 0.89, p < 0.001). Bland-Altman analysis identified bias in 64mT segmentations (mean = 1.6 ml, standard error = 5.2 ml, limits of agreement = -19.0-15.9 ml), which over-estimated low lesion volume and under-estimated high volume (r = 0.74, p < 0.001). Visual inspection revealed over-segmentation was driven venous hyperintensities on 64mT T2-FLAIR. Lesion size drove segmentation accuracy, with 93% of lesions > 1.0 ml and all lesions > 1.5 ml being detected. Using multi-acquisition volume averaging, we were able to generate 1.6 mm isotropic images on the 64mT device. Overall, our results demonstrate that in established MS, a portable 64mT MRI scanner can identify white matter lesions, and that automated estimates of total lesion volume correlate with measurements from 3T scans.

Identification of a polymorphism in the N gene of SARS-CoV-2 that adversely impacts detection by a widely-used RT-PCR assay

We identify a mutation in the N gene of SARS-CoV-2 that adversely affects annealing of a commonly used RT-PCR primer; epidemiologic evidence suggests the virus retains pathogenicity and competence for spread. This reinforces the importance of using multiple targets, preferably in at least 2 genes, for robust SARS-CoV-2 detection. Article Summary LineA SARS-CoV-2 variant that occurs worldwide and has spread in California significantly affects diagnostic sensitivity of an N gene assay, highlighting the need to employ multiple viral targets for detection.

Recent developments and strategies for the discovery of TACE inhibitors.

INTRODUCTION: TNF-α plays a central role in certain autoimmune diseases as well as in inflammation. The current strategy for excluding TNF-α from circulation is to selectively inhibit TNF-α converting enzyme (TACE), an enzyme that cleaves mTNF-α to active TNF-α. Various TACE inhibitors have been discovered by using different strategies to control inflammatory diseases, cancer, and cardiac hypertrophy. AREAS COVERED: The present article summarizes the design and discovery of novel TACE inhibitors that have been reported in the literature since 2012 onwards. It also includes some reports concerning the new role that TACE plays in cancer and cardiac hypertrophy. EXPERT OPINION: So far, undertaken studies that have looked to design and develop small TACE inhibitors have been discouraging due to the failure of any TACE inhibitors to hit the market. However, some of the latest developments, such as with tartrate-based inhibitors, has given hope to the potentiality of a viable novel selective TACE inhibitor therapeutic in the future. Indeed, some of the novel peptidomimetics and monoclonal antibodies have great potential to pave the way for an effective and safe therapy by selectively inhibiting TACE enzyme.

Guidelines for medical management of nuclear/radiation emergencies.

Management of victim of radiation injury poses a wide spectrum of challenges to the health care provider starting with the evaluation of the damage, the kind of hospitalization and treatment and the regular monitoring of the patient. Undesirable clinical outcomes are probable if prodromal stage evolves rapidly and is severe. Critical systems like neurovascular, gastrointestinal, haematopoietic and cutaneous are afflicted in Acute Radiation Syndrome. Three main elements which are essential for assessment of prognosis and selection of treatment are vomiting onset time, kinetics of depletion of lymphocyte, and chromosome abnormalities. Larger incidents warrant, a well-structured national response system. Health care institutions must develop protocols to respond to radiation exposure related emergencies in tandem with the local response teams. Multidisciplinary approach between clinical specialists, nursing staff and psychological experts is of critical significance. External decontamination, estimation of dose and fluid and electrolyte replacements form part of support therapy. Reverse isolation, antacids, H2 blockers, use of reverse barrier nursing and prophylactic antimicrobials are part of the treatment plan. Patients with severe bone marrow damage will require blood products support. Increased recovery of neutrophils in radiationaccident victims is the rationale for the use of Colony Stimulating Factors. New directions are under evaluation which includes novel cytokine therapies like interleukin-7, keratinocyte growth factor, and thrombopoietin or its analogues. The final decision regarding allogenic haematopoietic stem cell transplant should be considered after considering the irradiation source, particularity of the situations or circumstances, associated injuries and disease.

Liver lobe graft side and outcomes in living-donor liver transplant with small-for-size grafts.

OBJECTIVES: Living-donor liver transplant with small-for-size grafts (graft-to-recipient weight ratio < 0.8) may provide satisfactory results. We compared outcomes between right and left donor lobe in living-donor liver transplant. MATERIALS AND METHODS: Patients who had living-donor liver transplant from 2006 to 2008 with graft-to-recipient weight ratio < 0.8 (graft: right lobe, 24 patients; left lobe, 26 patients) were reviewed retrospectively. RESULTS: There were no significant differences in demographic and preoperative clinical data between patients who received a right or left lobe liver graft. Duration of surgery was longer, cold ischemia time was shorter, and mean baseline portal vein flow was greater in transplants performed with left than right donor lobes. Portal vein flow modulation with splenectomy was performed when portal flow was > 250 mL/min/100 g graft. Small-for-size syndrome was observed in 6 recipients (14%), but no patient who developed small-for-size syndrome developed liver failure or required revision transplant. The frequency of small-for-size syndrome was significantly greater in patients who had left lobe (4 patients [15%]) than right lobe transplant (2 patients [8%]; P ≤ .05). Graft dysfunction-free survival was significantly greater with right than left lobe grafts. In multivariate analysis, graft side was the only significant risk factor for small-for-size syndrome. CONCLUSIONS: In patients having living-donor liver transplant with small-for-size grafts, outcome was better with right than left lobe grafts.

Population-based incidence of diabetes in Australian youth aged 10-18 yr: increase in type 1 diabetes but not type 2 diabetes.

BACKGROUND: Global incidence of childhood type 2 diabetes has increased, with a greater rise amongst certain ethnic groups. OBJECTIVES: To examine the change in the incidence of type 1 and type 2 diabetes in Australian youth, aged 10-18 yr, in New South Wales, Australia. METHODS: Prospective population-based incidence study (2001-2008). Primary case ascertainment was from the Australasian Paediatric Endocrine Group Diabetes Register, secondary independent ascertainment from the National Diabetes Register. RESULTS: There were 202 incident cases of type 2 diabetes (96 boys, 48%). The mean age at diagnosis (±SD) was 14.6 ± 2.5 yr; 93% were overweight (International Obesity Taskforce Grade ≥1). Mean HbA1c was 8.8 ± 2.8%. Ethnicity was Caucasian 31%, Indigenous Australian 20%, Southeast Asian 11%, North African/Middle Eastern 9%, and NewZealander/Melanesian/Polynesian 8%. The mean annual incidence of type 2 diabetes was 3.0 per 100 000 per year (95% confidence interval (CI): 2.6-3.4) and did not change over time. The mean annual incidence of type 1 diabetes was 22.0 per 100 000 per year (95% CI: 20.8-23.1), and increased by 3.8% per year [incidence rate ratio IRR: 1.04, 95% CI: 1.02-1.06, p = 0.001]. Incidence was higher in Indigenous vs. non-Indigenous youth, IRR: 6.9 (95% CI: 4.7-10.2, p < 0.001). CONCLUSION: In 10-18 yr old youth, in Australia, the incidence of type 2 diabetes has remained steady during the last decade; however, the incidence of type 1 diabetes continues to rise. Most common diabetes in Australian youth is type 1 diabetes.

Reconstructing single hepatic artery with two arterial stumps: biliary complications in pediatric living donor liver transplantation.

INTRODUCTION: Liver grafts can at times have two hepatic arterial stumps. This can result in a dilemma whether to reconstruct single or both the arteries. Hepatic artery (HA) thrombosis is the most dreaded complication in pediatric living donor liver transplantation (LDLT) as it can result in biliary complications and subsequent graft loss. We herein report the feasibility of reconstructing single hepatic artery in pediatric living donor liver transplantation having two arterial stumps in the liver graft. MATERIALS AND METHODS: From 2008 to 2010, 87 pediatric patients undergoing LDLT were divided into three groups. Group 1 (n = 20): two HA stumps with two HA reconstruction, Group 2 (n = 22): two HA stumps with one HA reconstruction and Group 3 (n = 45): one HA stump with one HA reconstruction. The decision regarding the reconstruction of single or multiple HAs was made depending on the pre-operative radiological and intraoperative assessments. RESULTS: The incidence of HA thrombosis (p = 0.126) and biliary complications (p = 0.617), was similar in the three groups. CONCLUSION: Single HA reconstruction does not increase the risk of biliary strictures in pediatric LDLT recipients having dual hepatic arterial stumps in the liver graft.

Elastography of soft materials and tissues by holographic imaging of surface acoustic waves.

We use optical interferometry to capture coherent surface acoustic waves for elastographic imaging. An inverse method is employed to convert multi-frequency data into an elastic depth profile. Using this method, we image elastic properties over a 55 mm range with <5 mm resolution. For relevance to breast cancer detection, we employ a tissue phantom with a tumor-like inclusion. Holographic elastography is also shown to be well-behaved in ex vivo tissue, revealing the subsurface position of a bone. Because digital holography can assess waves over a wide surface area, this constitutes a flexible new platform for large volume and non-invasive elastography.

Toward soft-tissue elastography using digital holography to monitor surface acoustic waves.

Measuring the elasticity distribution inside the human body is of great interest because elastic abnormalities can serve as indicators of several diseases. We present a method for mapping elasticity inside soft tissues by imaging surface acoustic waves (SAWs) with digital holographic interferometry. With this method, we show that SAWs are consistent with Rayleigh waves, with velocities proportional to the square root of the elastic modulus greater than 2-40 kPa in homogeneous tissue phantoms. In two-layer phantoms, the SAW velocity transitions approximately from that of the lower layer to that of the upper layer as frequency is increased in agreement with the theoretical relationship between SAW dispersion and the depth-dependent stiffness profile. We also observed deformation in the propagation direction of SAWs above a stiff inclusion placed 8 mm below the surface. These findings demonstrate the potential for quantitative digital holography-based elastography of soft tissues as a noninvasive method for disease detection.

RNA interference mediated silencing of Hsp60 gene in human monocytic myeloma cell line U937 revealed decreased dengue virus multiplication.

Heat shock proteins (Hsps) or stress proteins are highly conserved molecules and expressed in all cell types under stressful conditions like heat, cold, hypoxia and infections. The objective of the present study was to determine the effect of dengue virus infection on relative expression of stress proteins and their role in the progression of the infection. As macrophages are the primary host for dengue, human promonocytic myeloblastoma U937 cells were infected with dengue virus type 2 New Guinea C strain for the evaluation of Hsps expression. A significant expression of Hsp60 was observed in virally infected U937 cells as compared to controls. In order to determine the correlation between Hsp60 expression and viral multiplication in infected cells, expression of Hsp60 was down regulated by RNA interference. Viral multiplication was determined by quantification of viral RNA copy number using Real Time PCR and plaque formation assay in cellular supernatants of Hsp60 silenced cells. Intracellular quantification of viral load was also determined by flow cytometry. It was observed that down regulation of Hsp60 in virally infected cells resulted into decrease in viral RNA copy number, plaque forming units and intracellular viral load. At the same time down regulation also resulted in increased IFN-alpha level. These observations suggest that, elevated levels of Hsp60 expression in virally infected cells may help in viral multiplication and could be possible therapeutic targets for the management of dengue virus infection.

Effect of Hippophae rhamnoides leaf extract against Dengue virus infection in human blood-derived macrophages.

Dengue virus occurs as four distinct serotypes, called Dengue 1, 2, 3, and 4. Symptomatic dengue virus infection ranges from a self limited febrile illness, dengue fever (DF), to a more severe disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The anti-Dengue treatment is severely hampered as no specific therapeutic agents are available. Even present treatment strategies for Dengue are more supportive than curative. In the present study anti-dengue activity of Hippophae rhamnoides (Seabuckthorn, SBT) leaf extract was evaluated in Dengue virus type-2 infected blood-derived human macrophages as macrophages are the primary target of Dengue virus infection. Infected cells were treated with SBT leaf extract and compared with commercially available anti-viral drug, Ribavirin. The extract was able to maintain the cell viability of Dengue-infected cells at par with Ribavirin along with the decrease and increase in TNF-alpha and IFN-gamma respectively. Anti-dengue activity of SBT extract was further determined by the traditional plaque assay. These observations suggest that the SBT leaf extract has a significant anti-dengue activity and has the potential for the treatment of Dengue.

Effect of Seabuckthorn (Hippophae rhamnoides) flavone on immune system: an in-vitro approach.

There are several reports, which suggest that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavones, of Seabuckthorn (SBT) (Hippophae rhamnoides L.) fruit berry can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines. We have evaluated the immunomodulatory activity of ethanolic solution of SBT flavone (FLV) in human peripheral blood mononuclear cells (PBMCs). The SBT flavone was found to stimulate production of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in PBMCs. However, increased expressions of p-IkappaB, NF-kappaB, and p-p38 were found in flavone-treated human PBMCs with significantly suppressed expression of CD25 (IL-2R). There was no alteration found in the nitric oxide (NO) production in mouse macrophage cell line RAW 264.7. These observations suggest that stimulation of IL-6 and TNF-alpha secretion may contribute to the putative beneficial effects of dietary flavone against microbial infection.

Prostate cancer: genes, environment, immunity and the use of immunotherapy.

Prostate cancer remains the most prevalent noncutaneous cancer, leading to almost 30,000 deaths every year in men in the United States. A large body of knowledge emphasizes a strong influence of epidemiological factors such as lifestyle, environment and diet, on the development of prostate cancer. Although risk reduction of prostate cancer has been somewhat successful, effective prevention is still lacking. Immunotherapeutic approaches, although moderately complicated, remain promising in an effort to control the progression and development of the disease. Taken together, the parameters of epidemiological studies and immunotherapeutic regimens might eventually be the most effective and preventive approach for prostate cancer. This review highlights some of the events associated with the development and prevention of prostate cancer.

Aqueous extract of Rhodiola imbricata rhizome stimulates proinflammatory mediators via phosphorylated IkappaB and transcription factor nuclear factor-kappaB.

Modulation of immune response to alleviate diseases has long since been of interest. Plant extracts have been widely investigated for their possible immunomodulatory properties. We have evaluated the immunomodulatory activity of aqueous extract of Rhodiola rhizome in human peripheral blood mononuclear cells (PBMCs) and mouse macrophage cell line RAW 264.7. The Rhodiola extract was found to stimulate production of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in human PBMCs as well as RAW 264.7 cell line. It also increased production of nitric oxide synergistically in combination with lipopolysaccharide (LPS) in RAW 264.7. Rhodiola at 250 microg/ml increased the p-IkappaB expression in human PBMCs. Aqueous extract of Rhodiola (250 microg/ml) also activated the nuclear translocation of NF-kappaB in human PBMCs, which is comparable to the positive stimulant LPS. Thus, our present study suggests that Rhodiola most likely activates proinflammatory mediators via phosphorylated inhibitory kB and transcription factor NF-kB. Our study demonstrates immunostimulatory potential of aqueous extract of Rhodiola rhizome, that can be used for upregulation of immune response in patients with inadequate functioning of the immune system.