Contact potential difference of cellsin vitro as a measure of hydrophilic properties of the cell membrane

Construction of an apparatus for the determination of the cell contact potential difference and the measuring method are described. The contact potential differences are attributed to the degree of hydrophobic properties of the cell surface and referred to human erythrocytes and chloroplasts.     

The host inflammatory response contributes to disease severity in Crimean-Congo hemorrhagic fever virus infected mice

Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen. In cell culture, CCHFV is sensed by the cytoplasmic RNA sensor retinoic acid-inducible gene I (RIG-I) molecule and its adaptor molecule mitochondrial antiviral signaling (MAVS) protein. MAVS initiates both type I interferon (IFN-I) and proinflammatory responses. Here, we studied the role MAVS plays in CCHFV infection in mice in both the presence and absence of IFN-I activity. MAVS-deficient mice were not susceptible to CCHFV infection when IFN-I signaling was active and showed no signs of disease. When IFN-I signaling was blocked by antibody, MAVS-deficient mice lost significant weight, but were uniformly protected from lethal disease, whereas all control mice succumbed to infection. Cytokine activity in the infected MAVS-deficient mice was markedly blunted. Subsequent investigation revealed that CCHFV infected mice lacking TNF-α receptor signaling (TNFA-R-deficient), but not IL-6 or IL-1 activity, had more limited liver injury and were largely protected from lethal outcomes. Treatment of mice with an anti-TNF-α neutralizing antibody also conferred partial protection in a post-virus exposure setting. Additionally, we found that a disease causing, but non-lethal strain of CCHFV produced more blunted inflammatory cytokine responses compared to a lethal strain in mice. Our work reveals that MAVS activation and cytokine production both contribute to CCHFV pathogenesis, potentially identifying new therapeutic targets to treat this disease.     

Body temperature variation controls pre-mRNA processing and transcription of antiviral genes and SARS-CoV-2 replication

Antiviral innate immunity represents the first defense against invading viruses and is key to control viral infections, including SARS-CoV-2. Body temperature is an omnipresent variable but was neglected when addressing host defense mechanisms and susceptibility to SARS-CoV-2 infection. Here, we show that increasing temperature in a 1.5°C window, between 36.5 and 38°C, strongly increases the expression of genes in two branches of antiviral immunity, nitric oxide production and type I interferon response. We show that alternative splicing coupled to nonsense-mediated decay decreases STAT2 expression in colder conditions and suggest that increased STAT2 expression at elevated temperature induces the expression of diverse antiviral genes and SARS-CoV-2 restriction factors. This cascade is activated in a remarkably narrow temperature range below febrile temperature, which reflects individual, circadian and age-dependent variation. We suggest that decreased body temperature with aging contributes to reduced expression of antiviral genes in older individuals. Using cell culture and in vivo models, we show that higher body temperature correlates with reduced SARS-CoV-2 replication, which may affect the different vulnerability of children versus seniors toward severe SARS-CoV-2 infection. Altogether, our data connect body temperature and pre-mRNA processing to provide new mechanistic insight into the regulation of antiviral innate immunity.     

Physiologically based cord clamping for infants ≥32+0 weeks gestation: A randomised clinical trial and reference percentiles for heart rate and oxygen saturation for infants ≥35+0 weeks gestation

BackgroundGlobally, the majority of newborns requiring resuscitation at birth are full term or late-preterm infants. These infants typically have their umbilical cord clamped early (ECC) before moving to a resuscitation platform, losing the potential support of the placental circulation. Physiologically based cord clamping (PBCC) is clamping the umbilical cord after establishing lung aeration and holds promise as a readily available means of improving early newborn outcomes. In mechanically ventilated lambs, PBCC improved cardiovascular stability and reduced hypoxia. We hypothesised that PBCC compared to ECC would result in higher heart rate (HR) in infants needing resuscitation, without compromising safety.Methods and findingsBetween 4 July 2018 and 18 May 2021, infants born at ≥32⁺⁰ weeks’ gestation with a paediatrician called to attend were enrolled in a parallel-arm randomised trial at 2 Australian perinatal centres. Following initial stimulation, infants requiring further resuscitation were randomised within 60 seconds of birth using a smartphone-accessible web link. The intervention (PBCC) was to establish lung aeration, either via positive pressure ventilation (PPV) or effective spontaneous breathing, prior to cord clamping. The comparator was early cord clamping (ECC) prior to resuscitation. The primary outcome was mean HR between 60 to 120 seconds after birth, measured using 3-lead electrocardiogram, extracted from video recordings blinded to group allocation. Nonrandomised infants had deferred cord clamping (DCC) ≥120 seconds in the observational study arm.Among 508 at-risk infants enrolled, 123 were randomised (n = 63 to PBCC, n = 60 to ECC). Median (interquartile range, IQR) for gestational age was 39.9 (38.3 to 40.7) weeks in PBCC infants and 39.6 (38.4 to 40.4) weeks in ECC infants. Approximately 49% and 50% of the PBCC and ECC infants were female, respectively. Five infants (PBCC = 2, ECC = 3, 4% total) had missing primary outcome data. Cord clamping occurred at a median (IQR) of 136 (126 to 150) seconds in the PBCC arm and 37 (27 to 51) seconds in the ECC arm. Mean HR between 60 to 120 seconds after birth was 154 bpm (beats per minute) for PBCC versus 158 bpm for ECC (adjusted mean difference −6 bpm, 95% confidence interval (CI) −17 to 5 bpm, P = 0.39). Among 31 secondary outcomes, postpartum haemorrhage ≥500 ml occurred in 34% and 32% of mothers in the PBCC and ECC arms, respectively. Two hundred ninety-five nonrandomised infants (55% female) with median (IQR) gestational age of 39.6 (38.6 to 40.6) weeks received DCC. Data from these infants was used to create percentile charts of expected HR and oxygen saturation in vigorous infants receiving DCC. The trial was limited by the small number of infants requiring prolonged or advanced resuscitation. PBCC may provide other important benefits we did not measure, including improved maternal–infant bonding and higher iron stores.ConclusionsIn this study, we observed that PBCC resulted in similar mean HR compared to infants receiving ECC. The findings suggest that for infants ≥32⁺⁰ weeks’ gestation who receive brief, effective resuscitation at closely monitored births, PBCC does not provide additional benefit over ECC (performed after initial drying and stimulation) in terms of key physiological markers of transition. PBCC was feasible using a simple, low-cost strategy at both cesarean and vaginal births. The percentile charts of HR and oxygen saturation may guide clinicians monitoring the transition of at-risk infants who receive DCC.Trial registrationAustralian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12618000621213.

Shiraz Badurdeen and colleagues evaluate whether physiologically-based cord clamping provides physiological benefits over early cord clamping for infants requiring resuscitation at birth.     

Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats

Alternative lengthening of telomeres (ALT) occurs in ∼10% of cancer entities. However, little is known about the heterogeneity of ALT activity since robust ALT detection assays with high-throughput in situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate ALT activity in single cells from the accumulation of single-stranded telomeric DNA and RNA. It involves a one-step fluorescent in situ hybridization approach followed by fluorescence microscopy imaging. Our method reliably identified ALT in cancer cell lines from different tumor entities and was validated in three established models of ALT induction and suppression. Furthermore, we successfully applied ALT-FISH to spatially resolve ALT activity in primary tissue sections from leiomyosarcoma and neuroblastoma tumors. Thus, our assay provides insights into the heterogeneity of ALT tumors and is suited for high-throughput applications, which will facilitate screening for ALT-specific drugs.