Contact-FP: A Dimerization-Dependent Fluorescent Protein Toolkit for Visualizing Membrane Contact Site Dynamics.

Membrane contact sites (MCSs) are sites of close apposition between two organelles used to exchange ions, lipids, and information. Cells respond to changing environmental or developmental conditions by modulating the number, extent, or duration of MCSs. Because of their small size and dynamic nature, tools to study the dynamics of MCSs in live cells have been limited. Dimerization-dependent fluorescent proteins (ddFPs) targeted to organelle membranes are an ideal tool for studying MCS dynamics because they reversibly interact to fluoresce specifically at the interface between two organelles. Here, we build on previous work using ddFPs as sensors to visualize the morphology and dynamics of MCSs. We engineered a suite of ddFPs called Contact-FP that targets ddFP monomers to lipid droplets (LDs), the endoplasmic reticulum (ER), mitochondria, peroxisomes, lysosomes, plasma membrane, caveolae, and the cytoplasm. We show that these probes correctly localize to their target organelles. Using LDs as a test case, we demonstrate that Contact-FP pairs specifically localize to the interface between two target organelles. Titration of LD-mitochondria ddFPs revealed that these sensors can be used at high concentrations to drive MCSs or can be titrated down to minimally perturb and visualize endogenous MCSs. We show that Contact-FP probes can be used to: (1) visualize LD-mitochondria MCS dynamics, (2) observe changes in LD-mitochondria MCS dynamics upon overexpression of PLIN5, a known LD-mitochondrial tether, and (3) visualize two MCSs that share one organelle simultaneously (e.g., LD-mitochondria and LD-ER MCSs). Contact-FP probes can be optimized to visualize MCSs between any pair of organelles represented in the toolkit.

PLIN5 interacts with FATP4 at membrane contact sites to promote lipid droplet-to-mitochondria fatty acid transport.

Cells adjust their metabolism by remodeling membrane contact sites that channel metabolites to different fates. Lipid droplet (LD)-mitochondria contacts change in response to fasting, cold exposure, and exercise. However, their function and mechanism of formation have remained controversial. We focused on perilipin 5 (PLIN5), an LD protein that tethers mitochondria, to probe the function and regulation of LD-mitochondria contacts. We demonstrate that efficient LD-to-mitochondria fatty acid (FA) trafficking and ß-oxidation during starvation of myoblasts are promoted by phosphorylation of PLIN5 and require an intact PLIN5 mitochondrial tethering domain. Using human and murine cells, we further identified the acyl-CoA synthetase, FATP4 (ACSVL4), as a mitochondrial interactor of PLIN5. The C-terminal domains of PLIN5 and FATP4 constitute a minimal protein interaction capable of inducing organelle contacts. Our work suggests that starvation leads to phosphorylation of PLIN5, lipolysis, and subsequent channeling of FAs from LDs to FATP4 on mitochondria for conversion to fatty-acyl-CoAs and subsequent oxidation.

Population study on diagnosis, treatment and outcomes of critically ill patients with tuberculosis (2008-2018).

BACKGROUND: Tuberculosis (TB) is a preventable and curable disease, but mortality remains high among those who develop sepsis and critical illness from TB. METHODS: This was a population-based, multicentre retrospective cohort study of patients admitted to all 15 publicly funded Hong Kong adult intensive care units (ICUs) between 1 April 2008 and 31 March 2019. 940 adult critically ill patients with at least one positive Mycobacterium tuberculosis (MTB) culture were identified out of 133 858 ICU admissions. Generalised linear modelling was used to determine the impact of delay in TB treatment on hospital mortality. Trend of annual Acute Physiology and Chronic Health Evaluation (APACHE) IV-adjusted standardised mortality ratio (SMR) over the 11-year period was analysed by Mann-Kendall's trend test. RESULTS: ICU and hospital mortality were 24.7% (232/940) and 41.1% (386/940), respectively. Of those who died in the ICU, 22.8% (53/232) never received antituberculosis drugs. SMR for ICU patients with TB remained unchanged over the study period (Kendall's τb=0.37, p=0.876). After adjustment for age, Charlson comorbidity index, APACHE IV, albumin, vasopressors, mechanical ventilation and renal replacement therapy, delayed TB treatment was directly associated with hospital mortality. In 302/940 (32.1%) of patients, TB could only be established from MTB cultures alone as Ziehl-Neelsen staining or PCR was either not performed or negative. Among this group, only 31.1% (94/302) had concurrent MTB PCR performed. CONCLUSIONS: Survival of ICU patients with TB has not improved over the last decade and mortality remains high. Delay in TB treatment was associated with higher hospital mortality. Use of MTB PCR may improve diagnostic yield and facilitate early treatment.

Oocyte Development and Quality in Young and Old Mice following Exposure to Atrazine.

BACKGROUND: Egg development has unique features that render it vulnerable to environmental perturbation. The herbicide atrazine is an endocrine disruptor shown to have detrimental effects on reproduction across several vertebrate species. OBJECTIVES: This study was designed to determine whether exposure to low levels of atrazine impairs meiosis in female mammals, using a mouse model; in particular, the study's researchers sought to determine whether and how the fidelity of oocyte chromosome segregation may be affected and whether aging-related aneuploidy is exacerbated. METHODS: Female C57BL/6J mice were exposed to two levels of atrazine in drinking water: The higher level equaled aqueous saturation, and the lower level corresponded to detected environmental contamination. To model developmental exposure, atrazine was ingested by pregnant females at 0.5 d post coitum and continued until pups were weaned at 21 d postpartum. For adult exposure, 2-month-old females ingested atrazine for 3 months. Following exposure, various indicators of oocyte development and quality were determined, including: a) chromosome synapsis and crossing over in fetal oocytes using immunofluorescence staining of prophase-I chromosome preparations; b) sizes of follicle pools in sectioned ovaries; c) efficiencies of in vitro fertilization and early embryogenesis; d) chromosome alignment and segregation in cultured oocytes; e) chromosomal errors in metaphase-I and -II (MI and MII) preparations; and f) sister-chromatid cohesion via immunofluorescence intensity of cohesin subunit REC8 on MI-chromosome preparations, and measurement of interkinetochore distances in MII preparations. RESULTS: Mice exposed to atrazine during development showed slightly higher levels of defects in chromosome synapsis, but sizes of initial follicle pools were indistinguishable from controls. However, although more eggs were ovulated, oocyte quality was lower. At the chromosome level, frequencies of spindle misalignment and numerical and structural abnormalities were greater at both meiotic divisions. In vitro fertilization was less efficient, and there were more apoptotic cells in blastocysts derived from eggs of atrazine-exposed females. Similar levels of chromosomal defects were seen in oocytes following both developmental and adult exposure regimens, suggesting quiescent primordial follicles may be a consequential target of atrazine. An important finding was that defects were observed long after exposure was terminated. Moreover, chromosomally abnormal eggs were very frequent in older mice, implying that atrazine exposure during development exacerbates effects of maternal aging on oocyte quality. Indeed, analogous to the effects of maternal age, weaker cohesion between sister chromatids was observed in oocytes from atrazine-exposed animals. CONCLUSION: Low-level atrazine exposure caused persistent changes to the female mammalian germline in mice, with potential consequences for reproductive lifespan and congenital disease. https://doi.org/10.1289/EHP11343.

Critically ill patients with COVID-19 in Hong Kong: a multicentre retrospective observational cohort study

Objective: To report the first eight cases of critically ill patients with coronavirus disease 2019 (COVID-19) in Hong Kong, describing the treatments and supportive care they received and their 28-day outcomes. Design: Multicentre retrospective observational cohort study. Setting: Three multidisciplinary intensive care units (ICUs) in Hong Kong. Participants: All adult critically ill patients with confirmed COVID-19 admitted to ICUs in Hong Kong between 22 January and 11 February 2020. Main outcome measure: 28-day mortality. Results: Eight out of 49 patients with COVID-19 (16%) were admitted to Hong Kong ICUs during the study period. The median age was 64.5 years (range, 42­70) with a median admission Sequential Organ Failure Assessment (SOFA) score of 6 (IQR, 4­7). Six patients (75%) required mechanical ventilation, six patients (75%) required vasopressors and two (25%) required renal replacement therapy. None of the patients required prone ventilation, nitric oxide or extracorporeal membrane oxygenation. The median times to shock reversal and extubation were 9 and 11 days respectively. At 28 days, one patient (12%) had died and the remaining seven (88%) all survived to ICU discharge. Only one of the survivors (14%) still required oxygen at 28 days. Conclusion: Critically ill patients with COVID-19 often require a moderate duration of mechanical ventilation and vasopressor support. Most of these patients recover and survive to ICU discharge with supportive care using lung protective ventilation strategies, avoiding excess fluids, screening and treating bacterial co-infection, and timely intubation. Lower rather than upper respiratory tract viral burden correlates with clinical severity of illness.

Increased vestibular contribution to posture control in individuals with chronic headache.

OBJECTIVE: Numerous studies have identified an association between headache disorders and vestibular symptoms, such as dizziness, vertigo, and motion sensitivity. Using bipolar, binaural galvanic vestibular stimulation (GVS), our objectives were to (1) determine the degree of vestibular sensitivity in borderline headache sufferers, and (2) characterize the postural response to vestibular perturbation in these individuals. METHODS: Fourteen volunteers participated in this study: 8 individuals with chronic headache (6 with migraine and 2 with tension type headache (TTH)), and 6 healthy control subjects. Thirty trials of 15-second duration were conducted across 6 conditions (GVS left (L) or right (R), or no stimulation (No GVS) with either eyes open (EO) or eyes closed (EC)). Peak medial-lateral (M-L) centre of pressure (CoP) was calculated during quiet stance. RESULTS: Headache subjects demonstrated significantly higher peak displacement of the M-L CoP than control subjects (p=0.0461). Although peak M-L CoP in both the EO (p = 0.0753) and EC (p=0.09623) visual conditions were not significantly different between headache sufferers compared to healthy controls, there is some suggestion that people with chronic headache may use vestibular information to a greater extent and that the presence of vision may not sufficiently compensate for vestibular induced instability. The initial push M-L CoP was not significantly different between groups, suggesting different sensory contributions for the initial and latter response to GVS. CONCLUSIONS: People with chronic headache exhibit increased postural sway, which may reflect the re-weighting of sensory information with an increased vestibular and a reduced visual contribution to postural control. SIGNIFICANCE: These results support existing research on vestibular abnormalities in chronic headache sufferers that may provide a basis for future treatment therapies.