Single-Molecule Super-Resolution Imaging of T-Cell Plasma Membrane CD4 Redistribution upon HIV-1 Binding

The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4⁺ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4⁺ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For one of the first times, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host CD4⁺ T cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function.     

Application of Super-Resolution and Advanced Quantitative Microscopy to the Spatio-Temporal Analysis of Influenza Virus Replication

With an estimated three to five million human cases annually and the potential to infect domestic and wild animal populations, influenza viruses are one of the greatest health and economic burdens to our society, and pose an ongoing threat of large-scale pandemics. Despite our knowledge of many important aspects of influenza virus biology, there is still much to learn about how influenza viruses replicate in infected cells, for instance, how they use entry receptors or exploit host cell trafficking pathways. These gaps in our knowledge are due, in part, to the difficulty of directly observing viruses in living cells. In recent years, advances in light microscopy, including super-resolution microscopy and single-molecule imaging, have enabled many viral replication steps to be visualised dynamically in living cells. In particular, the ability to track single virions and their components, in real time, now allows specific pathways to be interrogated, providing new insights to various aspects of the virus-host cell interaction. In this review, we discuss how state-of-the-art imaging technologies, notably quantitative live-cell and super-resolution microscopy, are providing new nanoscale and molecular insights into influenza virus replication and revealing new opportunities for developing antiviral strategies.     

Effect of combined AT1 receptor and aldosterone receptor antagonism on plasminogen activator inhibitor-1

Aldosterone enhances angiotensin II (Ang II)-induced plasminogen activator inhibitor (PAI)-1 expression in vitro. This study tested the hypothesis that angiotensin II type 1 (AT(1)) and aldosterone receptor antagonism interact to decrease PAI-1 in humans. Effects of candesartan (16 mg/d), spironolactone (25 mg/d), or combined candesartan/spironolactone on mean arterial pressure (MAP), endocrine, and fibrinolytic variables were measured in 18 normotensive subjects [age 33.7 yr (95% confidence interval 29.3, 38.0), body mass index 26.6 (24.7, 28.4) kg/m(2)] in whom the renin-angiotensin-aldosterone system was activated by furosemide (20 mg/d). Candesartan [83.3 mm Hg (78.9, 87.7)], but not spironolactone [89.4 mm Hg (85.4, 93.5)], decreased MAP, compared with baseline [92.2 mm Hg (88.9, 95.5), P < 0.001] and furosemide alone [89.1 mm Hg (85.7, 92.4), P = 0.002]. Coadministration of spironolactone with candesartan did not further decrease MAP. Candesartan dramatically increased Ang II [177.9 pg/ml (113.3, 242.6)], compared with baseline [34.8 pg/ml (29.3, 40.4), P = 0.002] and furosemide alone [40.6 pg/ml (29.7, 51.5), P = 0.003]. Spironolactone increased Ang II [51.5 pg/ml (41.3, 61.7), P = 0.014 vs. baseline, P = 0.004 vs. candesartan]. There was no additive effect of candesartan and spironolactone on Ang II [197.6 pg/ml (134.2, 261.0)]. Aldosterone was lower during candesartan [8.9 ng/dl (7.3, 10.6), P = 0.007] than during furosemide alone [14.1 ng/dl (10.9, 17.3), P = 0.007], spironolactone [18.7 ng/dl (14.5, 22.9), P = 0.002], or combined candesartan/spironolactone [13.9 ng/dl (11.8, 15.9), P = 0.006]. Furosemide increased PAI-1 antigen [27.8 ng/ml (20.6, 35.0), P = 0.002 vs. 19.3 ng/ml (13.4, 25.2) baseline], even in the presence of candesartan [27.2 ng/ml (16.5, 37.8), P = 0.042 vs. baseline] or spironolactone [27.3 ng/ml (17.9, 36.8), P = 0.015 vs. baseline]. However, coadministration of AT(1) and aldosterone receptor antagonists prevented the furosemide-induced increase in PAI-1 [19.2 ng/ml (9.8, 28.6), P = 0.974 vs. baseline, P < 0.05 vs. candesartan, spironolactone or furosemide alone]. This study evidences an interactive effect of endogenous Ang II and aldosterone on PAI-1 production in humans.     

Comparison of clindamycin, rifampin, tetracycline, metronidazole, and penicillin for efficacy in prevention of experimental gas gangrene due to Clostridium perfringens

Gas gangrene caused by Clostridium perfringens is associated with significant mortality and morbidity in spite of penicillin treatment. Although prompt surgical debridement has been established as the primary therapeutic objective, additional studies are needed for determination of the optimal antimicrobial therapy. In a mouse model of gas gangrene caused by Clostridium perfringens, clindamycin, metronidazole, rifampin, and tetracycline were all more efficacious than penicillin (P less than .05). Survival of penicillin-treated mice was not significantly better than that of untreated controls in spite of serum levels that ranged up to 77-1,800 micrograms/ml. Responses to metronidazole were highly dose dependent. For example, 60% of mice survived after 75 mg of metronidazole/kg, but only 10% survived after 19 mg/kg. In contrast, clindamycin was highly effective over a broad dosing range (8.6-86 mg/kg). The efficacy of all antibiotics was reduced if treatment was delayed or larger inocula of bacteria were used.     

Biomechanics of milk extraction during breast-feeding

How do infants extract milk during breast-feeding? We have resolved a century-long scientific controversy, whether it is sucking of the milk by subatmospheric pressure or mouthing of the nipple–areola complex to induce a peristaltic-like extraction mechanism. Breast-feeding is a dynamic process, which requires coupling between periodic motions of the infant’s jaws, undulation of the tongue, and the breast milk ejection reflex. The physical mechanisms executed by the infant have been intriguing topics. We used an objective and dynamic analysis of ultrasound (US) movie clips acquired during breast-feeding to explore the tongue dynamic characteristics. Then, we developed a new 3D biophysical model of the breast and lactiferous tubes that enables the mimicking of dynamic characteristics observed in US imaging during breast-feeding, and thereby, exploration of the biomechanical aspects of breast-feeding. We have shown, for the first time to our knowledge, that latch-on to draw the nipple–areola complex into the infant mouth, as well as milk extraction during breast-feeding, require development of time-varying subatmospheric pressures within the infant’s oral cavity. Analysis of the US movies clearly demonstrated that tongue motility during breast-feeding was fairly periodic. The anterior tongue, which is wedged between the nipple–areola complex and the lower lips, moves as a rigid body with the cycling motion of the mandible, while the posterior section of the tongue undulates in a pattern similar to a propagating peristaltic wave, which is essential for swallowing.Breast-feeding is strongly publicized and encouraged by many societies and communities. It is well accepted that breast milk provides the infant both nutrients and immunities required for growth and development during the first months after birth. It is less known that breast-fed infants exercise and prepare their orofacial muscles for future tasks of speaking and chewing (1), and also have higher oxygen saturation than bottle-fed infants (2). Breast-feeding is the outcome of a dynamic synchronization between oscillation of the infant’s mandible, rhythmic motility of the tongue, and the breast milk ejection reflex that drives maternal milk toward the nipple outlet. First, the infant latches onto the breast and nipple so that the nipple, areola, and underlying mammary tissue and lactiferous ducts are drawn into the infant’s mouth with the nipple tip extended as far as the hard–soft palate junction (HSPJ). Then, the infant moves its mandible up and down, compressing the areola and the underlying lactiferous ducts with its gums in a suckling process that extracts the milk into its mouth (3, 4). Simultaneous with compression, spontaneous undulating motions of the infant tongue channel the milk posteriorly and trigger the swallowing reflex (5). During breast-feeding, suckling, swallowing, and breathing are coordinated by the central nervous system in a way that allows for the infant’s continuous feeding without breathing interruptions (2, 6, 7).The physical mechanisms that enable the infant to extract milk from the breast have intrigued scientists for more than a century (8). The two proposed mechanisms that have been a subject of scientific controversy to this day are (i) sucking—emptying of the nipple–breast contents by development of subatmospheric pressures within the infant oral cavity (9–12) and (ii) mouthing—squeezing out of the nipple–areola contents by compression between the jaws or other mouth parts (3). With the appearance of cine–X-ray and ultrasound (US) imaging modalities, a significant role was also attributed to tongue undulation which was naturally referred to as “tongue peristalsis” while chewing the nipple (13, 14). However, advanced computational modeling has not yet been used along with imaging data to perform hypothesis testing on the underlying explanations of the suckling behavior during breast-feeding.We have explored the physical aspects of infant feeding via noninvasive visualizations of the moving components in the oral cavity and a biophysical model. An objective dynamic analysis of submental US imaging of the midsagittal cross-section of the oral cavity during infant feeding was used to study the dynamic characteristics of tongue motion with respect to the rigid upper palate. A 3D fluid–structure interaction (FSI) biophysical model was developed to simulate milk extraction during breast-feeding.     

The degree of fetal metformin exposure does not influence fetal outcome in gestational diabetes mellitus

The purpose of the study was to examine in vivo placental transfer of metformin, its association with neonatal outcome in metformin-treated gestational diabetes (GDM) patients, and influence of metformin exposure on maternal glycemic control and weight gain. Two hundred and seventeen GDM patients were randomized to metformin or insulin in Turku University Hospital, Finland. Metformin concentrations were determined by mass spectrometry in maternal serum at 36 gestational weeks (gw) and at birth, and in umbilical cord blood. Main outcome measures were birth weight, gw at birth, umbilical artery pH and neonatal hypoglycemia, maternal weight gain, HbA1c and fructosamine concentration. Median umbilical cord/maternal serum metformin concentration ratio was 0.73. There were no differences in birth weight measured in grams or SD units (p = 0.49), or gw at birth (p always ≥0.49) between insulin- and metformin-treated patients stratified by trough metformin concentration tertiles measured at 36 gw. Rate of neonatal hypoglycemia (p = 0.92) and umbilical artery pH value (p = 0.78) was similar in insulin- and metformin-treated patients stratified by cord metformin concentration tertiles. Maternal glycemic control was similar in metformin concentration tertiles at 36 gw. Maternal weight gain was 223 g greater per week (p = 0.038) in the lowest metformin tertile compared to other tertiles combined. Maternal and fetal exposure to metformin is similar. Maternal or fetal metformin concentrations do not predict maternal glycemic control or neonatal outcome, but low maternal exposure may lead to greater maternal weight gain.