The therapeutically actionable long non-coding RNA 'T-RECS' is essential to cancer cells' survival in NRAS/MAPK-driven melanoma.

Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impact on normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.

Comparison of Differences in Cohort (Forward) and Case Control (Backward) Methodologic Approaches for Validation of the Hypotension Prediction Index.

BACKGROUND: The Hypotension Prediction Index (the index) software is a machine learning algorithm that detects physiologic changes that may lead to hypotension. The original validation used a case control (backward) analysis that has been suggested to be biased. This study therefore conducted a cohort (forward) analysis and compared this to the original validation technique. METHODS: A retrospective analysis of data from previously reported studies was conducted. All data were analyzed identically with two different methodologies, and receiver operating characteristic curves were constructed. Both backward and forward analyses were performed to examine differences in area under the receiver operating characteristic curves for the Hypotension Prediction Index and other hemodynamic variables to predict a mean arterial pressure (MAP) less than 65 mmHg for at least 1 min 5, 10, and 15 min in advance. RESULTS: The analysis included 2,022 patients, yielding 4,152,124 measurements taken at 20-s intervals. The area under the curve for the index predicting hypotension analyzed by backward and forward methodologies respectively was 0.957 (95% CI, 0.947 to 0.964) versus 0.923 (95% CI, 0.912 to 0.933) 5 min in advance, 0.933 (95% CI, 0.924 to 0.942) versus 0.923 (95% CI, 0.911 to 0.933) 10 min in advance, and 0.929 (95% CI, 0.918 to 0.938) versus 0.926 (95% CI, 0.914 to 0.937) 15 min in advance. No variable other than MAP had an area under the curve greater than 0.7. The areas under the curve using forward analysis for MAP predicting hypotension 5, 10, and 15 min in advance were 0.932 (95% CI, 0.920 to 0.940), 0.929 (95% CI, 0.918 to 0.938), and 0.932 (95% CI, 0.921 to 0.940), respectively. The R2 for the variation in the index due to MAP was 0.77. CONCLUSIONS: Using an updated methodology, the study found that the utility of the Hypotension Prediction Index to predict future hypotensive events is high, with an area under the receiver operating characteristics curve similar to that of the original validation method.

The effect of liver surgery and fluid strategy on renin activity and aldosterone and anti-diuretic hormone levels: a secondary analysis of the GALILEO trial.

BACKGROUND: It is unknown whether liver surgery leads to increased RAAS activity and anti-diuretic hormone (ADH) levels and subsequent fluid accumulation. Furthermore, it is unknown whether the peri-operative fluid strategy changes this effect. METHODS: This is a pre-planned post hoc analysis of a randomised controlled trial which compared restrictive (n = 20) versus liberal fluid strategy (n = 20) in patients undergoing liver surgery. Primary outcomes for the current study were the difference in hormone levels after anaesthesia induction and after liver resection. Fluid overload was defined as a ≥10% increase in weight. RESULTS: Renin activity (6 [2.1-15.5] vs. 12 [4.6-33.5]) and ADH levels (6.0 [1.7-16.3] vs. 3.8 [1.6-14.7]) did not differ significantly before and after resection. However, aldosterone levels were significantly higher after resection (0.30 [0.17-0.49] vs. 0.69 [0.31-1.21] ). Renin activity and aldosterone levels did not differ between the groups. ADH was significantly higher in the restrictive strategy group (1.6 [1.1-2.1] vs 5.9 [3.8-16.0]). No differences in hormone levels were found in patients with and without fluid overload. DISCUSSION: Aldosterone levels increased after liver surgery but renin activity and ADH levels did not. ADH levels were higher in the restrictive group. Development of post-operative fluid overload was not associated with RAAS activity or ADH levels.

Insight into the molecular basis of substrate recognition by the wall teichoic acid glycosyltransferase TagA.

Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and ß-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding. Here, we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-N-acetyl-d-mannosamine, revealing the molecular basis of substrate binding. Native MS experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the C-terminal polypeptide tail facilitates catalysis by encapsulating the UDP-N-acetyl-d-mannosamine substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data, we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.

CYP26B1-related disorder: expanding the ends of the spectrum through clinical and molecular evidence.

CYP26B1 metabolizes retinoic acid in the developing embryo to regulate its levels. A limited number of individuals with pathogenic variants in CYP26B1 have been documented with a varied phenotypic spectrum, spanning from a severe manifestation involving skull anomalies, craniosynostosis, encephalocele, radio-humeral fusion, oligodactyly, and a narrow thorax, to a milder presentation characterized by craniosynostosis, restricted radio-humeral joint mobility, hearing loss, and intellectual disability. Here, we report two families with CYP26B1-related phenotypes and describe the data obtained from functional studies of the variants. Exome and Sanger sequencing were used for variant identification in family 1 and family 2, respectively. Family 1 reflects a mild phenotype, which includes craniofacial dysmorphism with brachycephaly (without craniosynostosis), arachnodactyly, reduced radioulnar joint movement, conductive hearing loss, learning disability-and compound heterozygous CYP26B1 variants: (p.[(Pro118Leu)];[(Arg234Gln)]) were found. In family 2, a stillborn fetus presented a lethal phenotype with spina bifida occulta, hydrocephalus, poor skeletal mineralization, synostosis, limb defects, and a synonymous homozygous variant in CYP26B1: c.1083C > A. A minigene assay revealed that the synonymous variant created a new splice site, removing part of exon 5 (p.Val361_Asp382del). Enzymatic activity was assessed using a luciferase assay, demonstrating a notable reduction in exogenous retinoic acid metabolism for the variant p.Val361_Asp382del. (~ 3.5 × decrease compared to wild-type); comparatively, the variants p.(Pro118Leu) and p.(Arg234Gln) demonstrated a partial loss of metabolism (1.7× and 2.3× reduction, respectively). A proximity-dependent biotin identification assay reaffirmed previously reported ER-resident protein interactions. Additional work into these interactions is critical to determine if CYP26B1 is involved with other biological events on the ER. Immunofluorescence assay suggests that mutant CYP26B1 is still localized in the endoplasmic reticulum. These results indicate that novel pathogenic variants in CYP26B1 result in varying levels of enzymatic activity that impact retinoic acid metabolism and relate to the distinct phenotypes observed.

Can volume-reduced plasma products prevent transfusion-associated circulatory overload in a two-hit animal model?

BACKGROUND AND OBJECTIVES: Transfusion-associated circulatory overload (TACO) is a pulmonary transfusion complication and a leading cause of transfusion-related morbidity and mortality. Volume overload and rising hydrostatic pressure as a consequence of transfusion are seen as the central pathway leading to TACO. A possible preventative measure for TACO could be the use of low-volume blood products like volume-reduced lyophilized plasma. We hypothesize that volume-reduced lyophilized plasma decreases circulatory overload leading to a reduced pulmonary capillary pressure and can therefore be an effective strategy to prevent TACO. MATERIALS AND METHODS: A validated two-hit animal model in rats with heart failure was used. Animals were randomized to receive 4 units of either solvent-detergent pooled plasma (SDP) as control, standard volume lyophilized plasma (LP-S) or hyperoncotic volume-reduced lyophilized plasma (LP-VR). The primary outcome was the difference between pre-transfusion and post-transfusion left ventricular end-diastolic pressure (ΔLVEDP). Secondary outcomes included markers for acute lung injury. RESULTS: LVEDP increased in all randomization groups following transfusion. The greatest elevation was seen in the group receiving LP-VR (+11.9 mmHg [5.9-15.6]), but there were no significant differences when compared to groups receiving either LP-S (+6.3 mmHg [2.9-13.4], p = 0.29) or SDP (+7.7 mmHg [4.5-10.5], p = 0.55). There were no significant differences in markers for acute lung injury, such as pulmonary wet/dry weight ratios, lung histopathology scores or PaO2 /FiO2 ratio between the three groups. CONCLUSION: Transfusion with hyperoncotic volume-reduced plasma did not attenuate circulatory overload compared to standard volume plasma and was therefore not an effective preventative strategy for TACO in this rat model.

Microbiological monitoring of coagulase-negative Staphylococcus in public drinking water fountains: Pathogenicity factors, antimicrobial resistance and potential health risks.

The presence of opportunistic bacteria such as coagulase-negative Staphylococcus (CoNS) in drinking water poses public health concerns because of its potential to cause human infection and due to its antimicrobial resistance (AMR) diversity. This study evaluated the occurrence, virulence markers and AMR of CoNS in 468 drinking water samples from 15 public fountains located in four urban parks of São Paulo city (Brazil). Out of 104 samples positive for the presence of Staphylococcus genus, we detected CoNS in 75 of them (16%), which did not meet the Brazilian sanitary standards for residual chlorine. All isolates were of concern to public health for being responsible for infection in humans from low to high severity, nine of them are considered the most of concern due to 63.6% being multiresistant to antimicrobials. The results demonstrated that CoNS in drinking water must not be neglected. It is concluded that the presence of resistant staphylococci in drinking water is a potential health risk, which urges feasible and quick control measures to protect human health, especially in crowded public places.

Dynamic cerebral autoregulation during step-wise increases in blood pressure during anaesthesia: A nonrandomised interventional trial.

BACKGROUND: Classically, cerebral autoregulation (CA) entails cerebral blood flow (CBF) remaining constant by cerebrovascular tone adapting to fluctuations in mean arterial pressure (MAP) between ∼60 and ∼150 mmHg. However, this is not an on-off mechanism; previous work has suggested that vasomotor tone is proportionally related to CA function. During propofol-based anaesthesia, there is cerebrovascular vasoconstriction, and static CA remains intact. Sevoflurane-based anaesthesia induces cerebral vasodilation and attenuates CA dose-dependently. It is unclear how this translates to dynamic CA across a range of blood pressures in the autoregulatory range. OBJECTIVE: The aim of this study was to quantify the effect of step-wise increases in MAP between 60 and 100 mmHg, using phenylephrine, on dynamic CA during propofol- and sevoflurane-based anaesthesia. DESIGN: A nonrandomised interventional trial. SETTING: Single centre enrolment started on 11 January 2019 and ended on 23 September 2019. PATIENTS: We studied American Society of Anesthesiologists (ASA) I/II patients undergoing noncardiothoracic, nonneurosurgical and nonlaparoscopic surgery under general anaesthesia. INTERVENTION: In this study, cerebrovascular tone was manipulated in the autoregulatory range by increasing MAP step-wise using phenylephrine in patients receiving either propofol- or sevoflurane-based anaesthesia. MAP and mean middle cerebral artery blood velocity (MCA Vmean ) were measured in ASA I and II patients, anaesthetised with either propofol ( n  = 26) or sevoflurane ( n  = 28), during 10 mmHg step-wise increments of MAP between 60 and 100 mmHg. Static CA was determined by plotting 2-min averaged MCA Vmean versus MAP. Dynamic CA was determined using transfer function analysis and expressed as the phase lead (°) between MAP and MCA Vmean oscillations, created with positive pressure ventilation with a frequency of 6 min -1 . MAIN OUTCOMES: The primary outcome of this study was the response of dynamic CA during step-wise increases in MAP during propofol- and sevoflurane-based anaesthesia. RESULTS: MAP levels achieved per step-wise increments were comparable between anaesthesia regiment (63 ±â€Š3, 72 ±â€Š2, 80 ±â€Š2, 90 ±â€Š2, 100 ±â€Š3 mmHg, and 61 ±â€Š4, 71 ±â€Š2, 80 ±â€Š2, 89 ±â€Š2, 98 ±â€Š4 mmHg for propofol and sevoflurane, respectively). MCA Vmean increased more during step-wise MAP increments for sevoflurane compared to propofol ( P ≤0.001). Dynamic CA improved during propofol (0.73° mmHg -1 , 95% CI 0.51 to 0.95; P  ≤ 0.001)) and less pronounced during sevoflurane-based anaesthesia (0.21°â€ŠmmHg -1 (95% CI 0.01 to 0.42, P  = 0.04). CONCLUSIONS: During general anaesthesia, dynamic CA is dependent on MAP, also within the autoregulatory range. This phenomenon was more pronounced during propofol anaesthesia than during sevoflurane. TRIAL REGISTRATION: NCT03816072 ( https://clinicaltrials.gov/ct2/show/NCT03816072 ).

Moxidectin and Ivermectin Inhibit SARS-CoV-2 Replication in Vero E6 Cells but Not in Human Primary Bronchial Epithelial Cells.

Antiviral therapies are urgently needed to treat and limit the development of severe COVID-19 disease. Ivermectin, a broad-spectrum anti-parasitic agent, has been shown to have anti-SARS-CoV-2 activity in Vero cells at a concentration of 5 µM. These limited in vitro results triggered the investigation of ivermectin as a treatment option to alleviate COVID-19 disease. However, in April 2021, the World Health Organization stated the following: "The current evidence on the use of ivermectin to treat COVID-19 patients is inconclusive." It is speculated that the in vivo concentration of ivermectin is too low to exert a strong antiviral effect. Here, we performed a head-to-head comparison of the antiviral activity of ivermectin and the structurally related, but metabolically more stable moxidectin in multiple in vitro models of SARS-CoV-2 infection, including physiologically relevant human respiratory epithelial cells. Both moxidectin and ivermectin exhibited antiviral activity in Vero E6 cells. Subsequent experiments revealed that these compounds predominantly act on the steps following virus cell entry. Surprisingly, however, in human-airway-derived cell models, both moxidectin and ivermectin failed to inhibit SARS-CoV-2 infection, even at concentrations of 10 µM. These disappointing results call for a word of caution in the interpretation of anti-SARS-CoV-2 activity of drugs solely based on their activity in Vero cells. Altogether, these findings suggest that even using a high-dose regimen of ivermectin, or switching to another drug in the same class, is unlikely to be useful for treatment of SARS-CoV-2 in humans.

Hypomorphic Mutations in TONSL Cause SPONASTRIME Dysplasia.

SPONASTRIME dysplasia is a rare, recessive skeletal dysplasia characterized by short stature, facial dysmorphism, and aberrant radiographic findings of the spine and long bone metaphysis. No causative genetic alterations for SPONASTRIME dysplasia have yet been determined. Using whole-exome sequencing (WES), we identified bi-allelic TONSL mutations in 10 of 13 individuals with SPONASTRIME dysplasia. TONSL is a multi-domain scaffold protein that interacts with DNA replication and repair factors and which plays critical roles in resistance to replication stress and the maintenance of genome integrity. We show here that cellular defects in dermal fibroblasts from affected individuals are complemented by the expression of wild-type TONSL. In addition, in vitro cell-based assays and in silico analyses of TONSL structure support the pathogenicity of those TONSL variants. Intriguingly, a knock-in (KI) Tonsl mouse model leads to embryonic lethality, implying the physiological importance of TONSL. Overall, these findings indicate that genetic variants resulting in reduced function of TONSL cause SPONASTRIME dysplasia and highlight the importance of TONSL in embryonic development and postnatal growth.

Autosomal-Recessive Mutations in MESD Cause Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) comprises a genetically heterogeneous group of skeletal fragility diseases. Here, we report on five independent families with a progressively deforming type of OI, in whom we identified four homozygous truncation or frameshift mutations in MESD. Affected individuals had recurrent fractures and at least one had oligodontia. MESD encodes an endoplasmic reticulum (ER) chaperone protein for the canonical Wingless-related integration site (WNT) signaling receptors LRP5 and LRP6. Because complete absence of MESD causes embryonic lethality in mice, we hypothesized that the OI-associated mutations are hypomorphic alleles since these mutations occur downstream of the chaperone activity domain but upstream of ER-retention domain. This would be consistent with the clinical phenotypes of skeletal fragility and oligodontia in persons deficient for LRP5 and LRP6, respectively. When we expressed wild-type (WT) and mutant MESD in HEK293T cells, we detected WT MESD in cell lysate but not in conditioned medium, whereas the converse was true for mutant MESD. We observed that both WT and mutant MESD retained the ability to chaperone LRP5. Thus, OI-associated MESD mutations produce hypomorphic alleles whose failure to remain within the ER significantly reduces but does not completely eliminate LRP5 and LRP6 trafficking. Since these individuals have no eye abnormalities (which occur in individuals completely lacking LRP5) and have neither limb nor brain patterning defects (both of which occur in mice completely lacking LRP6), we infer that bone mass accrual and dental patterning are more sensitive to reduced canonical WNT signaling than are other developmental processes. Biologic agents that can increase LRP5 and LRP6-mediated WNT signaling could benefit individuals with MESD-associated OI.

KIR2DL5B and HLA DRB1*12 alleles seems to be associated with protection against HIV-1 in serodiscordant couples in Burkina Faso.

The human immunodeficiency virus (HIV) belongs to the Retroviridae family and remains a public health problem in sub-Saharan Africa. Recent reports from WHO have shown that 33 million people died from HIV infections. HIV is one of the most serious fatal human diseases of the 20th and 21st centuries. However, variations in genetic and immunological factors are associated with protection against HIV infection in uninfected people exposed to HIV. This is the case with naturals killers which play an important role in the progression or regression of HIV infection. The objective of this study is to characterize certain HLA (human leukocyte antigen) class II genes and KIR genes in HIV-1 serodiscordant couples in Burkina Faso. This study was carried out at Burkina Faso among nineteen (19) HIV-1 serodiscordant couples. Classical multiplex PCR (SSP-PCR) was used to characterize the presence or absence of the KIR genes and certain class II HLAs (DRB1*11 and DRB1*12). The characterization of the KIR and HLA genes DRB1*11, DRB1*12 in this study demonstrated that the inhibitor KIR2DL5B, would confer protection against HIV-1 infection in seronegative partners (odd ratio [OR] = 0.13 [0.02-0.72] and p = 0.029), and the HLA DRB1*12 allele was associated with protection against HIV-1 infection in seronegative partners (OR = 0.16 [0.03-0.77] and p = 0.038). AA and Bx haplotypes were not found to be associated with HIV-1 infection in serodiscordant couples. This study confirms the involvement of the KIR genes in viral pathologies such as HIV-1 infection. Future larger-scale studies may provide a better understanding of the molecular mechanism by which the KIR haplotype and combination of KIR/HLA are associated with protection against HIV infection.

Autologous red blood cell transfusion does not result in a more profound increase in pulmonary capillary wedge pressure compared to saline in critically ill patients: A randomized crossover trial.

BACKGROUND AND OBJECTIVES: Transfusion-associated circulatory overload (TACO) is a major cause of severe transfusion-related morbidity. Transfusion of red blood cells (RBCs) has been shown to induce hydrostatic pressure overload. It is unclear which product-specific factors contribute. We set out to determine the effect of autologous RBC transfusion versus saline on pulmonary capillary wedge pressure (PCWP) change. MATERIALS AND METHODS: In a randomized crossover trial, patients who had undergone coronary bypass surgery were allocated to treatment post-operatively in the intensive care unit with either an initial 300 ml autologous RBC transfusion (salvaged during surgery) or 300 ml saline infusion first, followed by the other. Primary outcome was the difference in PCWP change. Secondary outcome measures were the difference in extra-vascular lung water index (EVLWI) and pulmonary vascular permeability index (PVPI). RESULTS: Change in PCWP was not higher after autologous RBC transfusion compared to saline (ΔPCWP 0.3 ± 0.4 vs. 0.1 ± 0.4 mmHg). ΔEVLWI and ΔPVPI were significantly decreased after autologous RBC transfusion compared to saline (ΔEVLWI -1.6 ± 0.6 vs. 0.2 ± 0.4, p = 0.02; ΔPVPI -0.3 ± 0.1 vs. 0.0 ± 0.1, p = 0.01). Haemodynamic variables and colloid osmotic pressure were not different for autologous RBC transfusion versus saline. CONCLUSION: Transfusion of autologous RBCs did not result in a more profound increase in PCWP compared to saline. RBC transfusion resulted in a decrease of EVLWI and PVPI compared to saline. Our data suggest that transfusing autologous RBCs may lead to less pulmonary oedema compared to saline. Future studies with allogeneic RBCs are needed to investigate other factors that may mediate the increase of PCWP, resulting in TACO.

Differential effects of speed and volume on transfusion-associated circulatory overload: A randomized study in rats.

BACKGROUND AND OBJECTIVES: Transfusion-associated circulatory overload (TACO) is the primary cause of transfusion-related mortality. Speed and volume of transfusion are major risk factors. The aim of this study was to investigate the interaction of red blood cell (RBC) transfusion speed and volume on the development of TACO. MATERIALS AND METHODS: A validated model for TACO in anaemic Lewis rats with an acute myocardial infarction was used. The effect on pulmonary hydrostatic pressure of one, two or four units of packed RBCs transfused in either 30 or 60 min was evaluated (3.3-26.6 ml·kg-1 ·hr-1 ). Pulmonary capillary pressure was measured as left ventricular end-diastolic pressure (LVEDP). Cardiac stress biomarkers atrial natriuretic-peptide (ANP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured 1-h post-transfusion. RESULTS: Thirty animals were included (n = 5 per group). Transfusion of RBCs increased LVEDP in a volume-dependent manner (ΔLVEDP [mmHg]: -0.95, +0.50, +6.26, p < 0.001). Fast transfusion increased overall ΔLVEDP by +3.5 mmHg and up to +11.8 mmHg in the four units' group (p = 0.016). Doubling transfusion speed increased ΔLVEDP more than doubling volume in the larger volume groups. No difference in ANP or NT-proBNP were seen in high transfusion volume or groups. CONCLUSION: Transfusion volume dose-dependently increased LVEDP, with speed of transfusion rapidly elevating LVEDP at higher transfusion volumes. ANP and NT-proBNP were not impacted by transfusion volume or speed in this model. TACO is seen as purely volume overload, however, this study emphasizes that limiting transfusion speed, as a modifiable risk factor, might aid in preventing TACO.

Performance of a machine-learning algorithm to predict hypotension in mechanically ventilated patients with COVID-19 admitted to the intensive care unit: a cohort study.

The Hypotension Prediction Index (HPI) is a commercially available machine-learning algorithm that provides warnings for impending hypotension, based on real-time arterial waveform analysis. The HPI was developed with arterial waveform data of surgical and intensive care unit (ICU) patients, but has never been externally validated in the latter group. In this study, we evaluated diagnostic ability of the HPI with invasively collected arterial blood pressure data in 41 patients with COVID-19 admitted to the ICU for mechanical ventilation. Predictive ability was evaluated at HPI thresholds from 0 to 100, at incremental intervals of 5. After exceeding the studied threshold, the next 20 min were screened for positive (mean arterial pressure (MAP) < 65 mmHg for at least 1 min) or negative (absence of MAP < 65 mmHg for at least 1 min) events. Subsequently, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and time to event were determined for every threshold. Almost all patients (93%) experienced at least one hypotensive event. Median number of events was 21 [7-54] and time spent in hypotension was 114 min [20-303]. The optimal threshold was 90, with a sensitivity of 0.91 (95% confidence interval 0.81-0.98), specificity of 0.87 (0.81-0.92), PPV of 0.69 (0.61-0.77), NPV of 0.99 (0.97-1.00), and median time to event of 3.93 min (3.72-4.15). Discrimination ability of the HPI was excellent, with an area under the curve of 0.95 (0.93-0.97). This validation study shows that the HPI correctly predicts hypotension in mechanically ventilated COVID-19 patients in the ICU, and provides a basis for future studies to assess whether hypotension can be reduced in ICU patients using this algorithm.

Molecular diagnosis in a cohort of 114 patients with rare skeletal dysplasias.

Molecular diagnosis is important to provide accurate genetic counseling of skeletal dysplasias (SD). Although next-generation sequencing (NGS) techniques are currently the preferred methods for analyzing these conditions, some of the published results have not shown a detection rate as high as it would be expected. The present study aimed to assess the diagnostic yield of targeted NGS combined with Sanger sequencing (SS) for low-coverage exons of genes of interest and exome sequencing (ES) in a series of patients with rare SD and use two patients as an example of our strategy. This study used two different in-house panels. Of 93 variants found in 88/114 (77%) patients, 57 are novel. The pathogenic variants found in the following genes: B3GALT6, PCYT1A, INPPL1, LIFR, of four patients were only detected by SS. In conclusion, the high diagnostic yield reached in the present study can be attributed to both a good selection of patients and the utilization of the SS for the insufficiently covered regions. Additionally, the two case reports-a patient with acrodysostosis related to PRKAR1A and another with ciliopathy associated with KIAA0753, add new and relevant clinical information to the current knowledge.

Serotonergic Drugs Inhibit Chikungunya Virus Infection at Different Stages of the Cell Entry Pathway.

Chikungunya virus (CHIKV) is an important reemerging human pathogen transmitted by mosquitoes. The virus causes an acute febrile illness, chikungunya fever, which is characterized by headache, rash, and debilitating (poly)arthralgia that can reside for months to years after infection. Currently, effective antiviral therapies and vaccines are lacking. Due to the high morbidity and economic burden in the countries affected by CHIKV, there is a strong need for new strategies to inhibit CHIKV replication. The serotonergic drug 5-nonyloxytryptamine (5-NT) was previously identified as a potential host-directed inhibitor for CHIKV infection. In this study, we determined the mechanism of action by which the serotonin receptor agonist 5-NT controls CHIKV infection. Using time-of-addition and entry bypass assays, we found that 5-NT predominantly inhibits CHIKV in the early phases of the replication cycle, at a step prior to RNA translation and genome replication. Intriguingly, however, no effect was seen during virus-cell binding, internalization, membrane fusion and genomic RNA (gRNA) release into the cell cytosol. In addition, we show that the serotonin receptor antagonist methiothepin mesylate (MM) also has antiviral properties toward CHIKV and specifically interferes with the cell entry process and/or membrane fusion. Taken together, pharmacological targeting of 5-HT receptors may represent a potent way to limit viral spread and disease severity.IMPORTANCE The rapid spread of mosquito-borne viral diseases in humans puts a huge economic burden on developing countries. For many of these infections, including those caused by chikungunya virus (CHIKV), there are no specific treatment possibilities to alleviate disease symptoms. Understanding the virus-host interactions that are involved in the viral replication cycle is imperative for the rational design of therapeutic strategies. In this study, we discovered an antiviral compound, elucidated its mechanism of action, and propose serotonergic drugs as potential host-directed antivirals for CHIKV.

Pathogenic variants in GNPTAB and GNPTG encoding distinct subunits of GlcNAc-1-phosphotransferase differentially impact bone resorption in patients with mucolipidosis type II and III.

PURPOSE: Pathogenic variants in GNPTAB and GNPTG, encoding different subunits of GlcNAc-1-phosphotransferase, cause mucolipidosis (ML) II, MLIII alpha/beta, and MLIII gamma. This study aimed to investigate the cellular and molecular bases underlying skeletal abnormalities in patients with MLII and MLIII. METHODS: We analyzed bone biopsies from patients with MLIII alpha/beta or MLIII gamma by undecalcified histology and histomorphometry. The skeletal status of Gnptgko and Gnptab-deficient mice was determined and complemented by biochemical analysis of primary Gnptgko bone cells. The clinical relevance of the mouse data was underscored by systematic urinary collagen crosslinks quantification in patients with MLII, MLIII alpha/beta, and MLIII gamma. RESULTS: The analysis of iliac crest biopsies revealed that bone remodeling is impaired in patients with GNPTAB-associated MLIII alpha/beta but not with GNPTG-associated MLIII gamma. Opposed to Gnptab-deficient mice, skeletal remodeling is not affected in Gnptgko mice. Most importantly, patients with variants in GNPTAB but not in GNPTG exhibited increased bone resorption. CONCLUSION: The gene-specific impact on bone remodeling in human individuals and in mice proposes distinct molecular functions of the GlcNAc-1-phosphotransferase subunits in bone cells. We therefore appeal for the necessity to classify MLIII based on genetic in addition to clinical criteria to ensure appropriate therapy.

A randomized, double-blind, phase 2b study to investigate the efficacy, safety, tolerability and pharmacokinetics of a single-dose regimen of ferroquine with artefenomel in adults and children with uncomplicated Plasmodium falciparum malaria.

BACKGROUND: For uncomplicated Plasmodium falciparum malaria, highly efficacious single-dose treatments are expected to increase compliance and improve treatment outcomes, and thereby may slow the development of resistance. The efficacy and safety of a single-dose combination of artefenomel (800 mg) plus ferroquine (400/600/900/1200 mg doses) for the treatment of uncomplicated P. falciparum malaria were evaluated in Africa (focusing on children ≤ 5 years) and Asia. METHODS: The study was a randomized, double-blind, single-dose, multi-arm clinical trial in patients aged > 6 months to < 70 years, from six African countries and Vietnam. Patients were followed up for 63 days to assess treatment efficacy, safety and pharmacokinetics. The primary efficacy endpoint was the polymerase chain reaction (PCR)-adjusted adequate clinical and parasitological response (ACPR) at Day 28 in the Per-Protocol [PP] Set comprising only African patients ≤ 5 years. The exposure-response relationship for PCR-adjusted ACPR at Day 28 and prevalence of kelch-13 mutations were explored. RESULTS: A total of 373 patients were treated: 289 African patients ≤ 5 years (77.5%), 64 African patients > 5 years and 20 Asian patients. None of the treatment arms met the target efficacy criterion for PCR-adjusted ACPR at Day 28 (lower limit of 95% confidence interval [CI] > 90%). PCR-adjusted ACPR at Day 28 [95% CI] in the PP Set ranged from 78.4% [64.7; 88.7%] to 91.7% [81.6; 97.2%] for the 400 mg to 1200 mg ferroquine dose. Efficacy rates were low in Vietnamese patients, ranging from 20 to 40%. A clear relationship was found between drug exposure (artefenomel and ferroquine concentrations at Day 7) and efficacy (primary endpoint), with higher concentrations of both drugs resulting in higher efficacy. Six distinct kelch-13 mutations were detected in parasite isolates from 10/272 African patients (with 2 mutations known to be associated with artemisinin resistance) and 18/20 Asian patients (all C580Y mutation). Vomiting within 6 h of initial artefenomel administration was common (24.6%) and associated with lower drug exposures. CONCLUSION: The efficacy of artefenomel/ferroquine combination was suboptimal in African children aged ≤ 5 years, the population of interest, and vomiting most likely had a negative impact on efficacy. Trial registration ClinicalTrials.gov, NCT02497612. Registered 14 Jul 2015, https://clinicaltrials.gov/ct2/show/NCT02497612?term=NCT02497612&draw=2&rank=1.

Effect of Hypotension Prediction Index-guided intraoperative haemodynamic care on depth and duration of postoperative hypotension: a sub-study of the Hypotension Prediction trial.

BACKGROUND: Intraoperative and postoperative hypotension are associated with morbidity and mortality. The Hypotension Prediction (HYPE) trial showed that the Hypotension Prediction Index (HPI) reduced the depth and duration of intraoperative hypotension (IOH), without excess use of intravenous fluid, vasopressor, and/or inotropic therapies. We hypothesised that intraoperative HPI-guided haemodynamic care would reduce the severity of postoperative hypotension in the PACU. METHODS: This was a sub-study of the HYPE study, in which 60 adults undergoing elective noncardiac surgery were allocated randomly to intraoperative HPI-guided or standard haemodynamic care. Blood pressure was measured using a radial intra-arterial catheter, which was connected to a FloTracIQ sensor. Hypotension was defined as MAP <65 mm Hg, and a hypotensive event was defined as MAP <65 mm Hg for at least 1 min. The primary outcome was the time-weighted average (TWA) of postoperative hypotension. Secondary outcomes were absolute incidence, area under threshold for hypotension, and percentage of time spent with MAP <65 mm Hg. RESULTS: Overall, 54/60 (90%) subjects (age 64 (8) yr; 44% female) completed the protocol, owing to failure of the FloTracIQ device in 6/60 (10%) patients. Intraoperative HPI-guided care was used in 28 subjects; 26 subjects were randomised to the control group. Postoperative hypotension occurred in 37/54 (68%) subjects. HPI-guided care did not reduce the median duration (TWA) of postoperative hypotension (adjusted median difference, vs standard of care: 0.118; 95% confidence interval [CI], 0-0.332; P=0.112). HPI-guidance reduced the percentage of time with MAP <65 mm Hg by 4.9% (adjusted median difference: -4.9; 95% CI, -11.7 to -0.01; P=0.046). CONCLUSIONS: Intraoperative HPI-guided haemodynamic care did not reduce the TWA of postoperative hypotension.