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Mesenchymal stromal cells (MSCs) are promising therapeutic agents for cartilage regeneration, including the potential of cells to promote chondrogenesis in vivo. However, process development and regulatory approval of MSCs as cell therapy products benefit from facile in vitro approaches that can predict potency for a given production run. Current standard in vitro approaches include a 21 day 3D differentiation assay followed by quantification of cartilage matrix proteins. We propose a novel biophysical marker that is cell population-based and can be measured from in vitro monolayer culture of MSCs. We hypothesized that the self-assembly pattern that emerges from collective-cell behavior would predict chondrogenesis motivated by our observation that certain features in this pattern, namely, topological defects, corresponded to mesenchymal condensations. Indeed, we observed a strong predictive correlation between the degree-of-order of the pattern at day 9 of the monolayer culture and chondrogenic potential later estimated from in vitro 3D chondrogenic differentiation at day 21. These findings provide the rationale and the proof-of-concept for using self-assembly patterns to monitor chondrogenic commitment of cell populations. Such correlations across multiple MSC donors and production batches suggest that self-assembly patterns can be used as a candidate biophysical attribute to predict quality and efficacy for MSCs employed therapeutically for cartilage regeneration., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Makhija et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two ER-associated lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. TMEM41B is also a recently validated host factor required by flaviviruses and coronaviruses. However, the exact underlying mechanism of TMEM41B in promoting viral infections remains an open question. Here, we validated that both TMEM41B and VMP1 are essential host dependency factors for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43), but not chikungunya virus (CHIKV). While HCoV-OC43 failed to replicate entirely in both TMEM41B- and VMP1-deficient cells, we detected diminished levels of DENV infections in these cell lines, which were accompanied by upregulation of the innate immune dsRNA sensors, RIG-I and MDA5. Nonetheless, this upregulation did not correspondingly induce the downstream effector TBK1 activation and Interferon-beta expression. Despite low levels of DENV replication, classical DENV replication organelles were undetectable in the infected TMEM41B-deficient cells, suggesting that the upregulation of the dsRNA sensors is likely a consequence of aberrant viral replication rather than a causal factor for reduced DENV infection. Intriguingly, we uncovered that the inhibitory effect of TMEM41B deficiency on DENV replication, but not HCoV-OC43, can be partially reversed using exogenous fatty acid supplements. In contrast, VMP1 deficiency cannot be rescued using the metabolite treatment. In line with the observed phenotypes, we found that both TMEM41B- and VMP1-deficient cells harbor higher levels of compromised mitochondria, especially in VMP1 deficiency which results in severe dysregulations of mitochondrial beta-oxidation. Using a metabolomic profiling approach, we revealed distinctive global dysregulations of the cellular metabolome, particularly lipidome, in TMEM41B- and VMP1-deficient cells. Our findings highlight a central role for TMEM41B and VMP1 in modulating multiple cellular pathways, including lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulations, to facilitate the replication of flaviviruses and coronaviruses., Competing Interests: The authors have declared that no competing interests exist.

Acute post-operative pain can remain untreated if patients cannot express themselves. The perfusion index (PI) may decrease when pain activates sympathetic tone and may increase after analgesics are administered. We evaluated if the perfusion index is a feasible indicator for objectively assessing pain relief in the postanesthesia care unit (PACU) and calculated the changes in PI measurements at the time of discharge from the PACU relative to baseline PI measurements to examine if the PI is a useful criterion for discharging patients from the postanesthesia care unit. This retrospective observational study enrolled female patients who were admitted for gynecological or general surgery. The patients received general anesthesia and were admitted to the postanesthesia care unit. The PI, visual analogue scale (VAS) score, heart rate, and blood pressure were recorded before and after administration of intravenous morphine. Changes in these parameters before and after analgesics were administered and the difference of these parameters between age and BMI subgroups were compared. The correlation between the PI and VAS score, ΔPI and ΔVAS, and %ΔPI and %ΔVAS were also evaluated. The percentage change in ΔPI (P9-T0/T0) of the patients at the time of discharge from the postanesthesia care unit relative to baseline PI measurements was calculated. Eighty patients were enrolled, and there were 123 instances during which analgesia was required. Heart rate, PI, and VAS score were significantly different before and after analgesics were administered (p < 0.0001). The difference of parameters between age and BMI subgroups were not significant. The correlation between the PI and VAS score, ΔPI and ΔVAS, and the percentage change in ΔPI and ΔVAS showed weak correlations in age, BMI subgroups, and all measurements. The baseline PI and the PI when arriving at and when being discharged from the postanesthesia care unit were significantly different (p < 0.01). The mean percentage change in Δ PI at the time of discharge from the PACU was 66.2%, and the 99% confidence interval was 12.2%~120.3%. The perfusion index was increased, and the VAS score was decreased significantly after analgesics were administered, but the correlation was weak in each subgroup. The VAS score is a subjective and psychometric parameter. The PI increased when partial pain relief was achieved after morphine was administered but did not reflect pain intensity or changes in the VAS score regardless of age or BMI. A percentage change in ΔPI at the time of discharge from the PACU relative to baseline PI measurements of greater than 12% can be used as a supplemental objective discharge criterion for pain assessment in the postanesthesia care unit.

Dengue virus (DENV) is the most prevalent arbovirus leading to an estimated 100 million symptomatic dengue infections every year. DENV can cause a spectrum of clinical manifestations, ranging from mild dengue fever (DF) to more life threatening forms such as dengue hemorrhagic fever (DHF). The clinical symptoms of DHF become evident typically at the critical phase of infection (5-7 days after onset of fever), yet the mechanisms that trigger transition from DF to DHF are not well understood. We performed a mass spectrometry-based metabolomic profiling of sera from adult DF and DHF patients at the critical and recovery phases of infection. There were 29 differentially expressed metabolites identified between DF and DHF at the critical phase. These include bile acids, purines, acylcarnitines, phospholipids, and amino acids. Bile acids were observed up to 5 fold higher levels among DHF compared to DF patients and were significantly correlated to the higher levels of aspartate transaminase (AST) and alanine transaminase (ALT), suggestive of liver injury among DHF. Uric acid, the most abundant antioxidant in the blood, was observed to be 1.5 fold lower among DHF compared to DF patients. This could result in decreased capacity of endogenous antioxidant defense and elevated oxidative stress among DHF patients. In the recovery phase, the levels of eight metabolites were still significantly higher or lower among DHF patients, including chenodeoxyglycocholic acid, one of the bile acids observed at the critical phase. This indicates potential prolonged adverse impact on the liver due to DENV infection in DHF patients. Our study identified altered metabolic pathways linked to DHF in the critical and recovery phases of dengue infection and provided insights into the different host and DENV interactions between DF and DHF. The results advance our understanding on the mechanisms of DHF pathogenesis, alluding to possible novel therapeutic targets to dengue management.

The genome sequence of V. cholerae O1 Biovar Eltor strain N16961 has revealed a putative antibiotic resistance (var) regulon that is predicted to encode a transcriptional activator (VarR), which is divergently transcribed relative to the putative resistance genes for both a metallo-β-lactamase (VarG) and an antibiotic efflux-pump (VarABCDEF). We sought to test whether these genes could confer antibiotic resistance and are organised as a regulon under the control of VarR. VarG was overexpressed and purified and shown to have β-lactamase activity against penicillins, cephalosporins and carbapenems, having the highest activity against meropenem. The expression of VarABCDEF in the Escherichia coli (ΔacrAB) strain KAM3 conferred resistance to a range of drugs, but most significant resistance was to the macrolide spiramycin. A gel-shift analysis was used to determine if VarR bound to the promoter regions of the resistance genes. Consistent with the regulation of these resistance genes, VarR binds to three distinct intergenic regions, varRG, varGA and varBC located upstream and adjacent to varG, varA and varC, respectively. VarR can act as a repressor at the varRG promoter region; whilst this repression was relieved upon addition of β-lactams, these did not dissociate the VarR/varRG-DNA complex, indicating that the de-repression of varR by β-lactams is indirect. Considering that the genomic arrangement of VarR-VarG is strikingly similar to that of AmpR-AmpC system, it is possible that V. cholerae has evolved a system for resistance to the newer β-lactams that would prove more beneficial to the bacterium in light of current selective pressures.

Effective triage of dengue patients early in the disease course for in- or out-patient management would be useful for optimal healthcare resource utilization while minimizing poor clinical outcome due to delayed intervention. Yet, early prognosis of severe dengue is hampered by the heterogeneity in clinical presentation and routine hematological and biochemical measurements in dengue patients that collectively correlates poorly with eventual clinical outcome. Herein, untargeted liquid-chromatography mass spectrometry metabolomics of serum from patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) in the febrile phase (<96 h) was used to globally probe the serum metabolome to uncover early prognostic biomarkers of DHF. We identified 20 metabolites that are differentially enriched (p<0.05, fold change >1.5) in the serum, among which are two products of tryptophan metabolism-serotonin and kynurenine. Serotonin, involved in platelet aggregation and activation decreased significantly, whereas kynurenine, an immunomodulator, increased significantly in patients with DHF, consistent with thrombocytopenia and immunopathology in severe dengue. To sensitively and accurately evaluate serotonin levels as prognostic biomarkers, we implemented stable-isotope dilution mass spectrometry and used convalescence samples as their own controls. DHF serotonin was significantly 1.98 fold lower in febrile compared to convalescence phase, and significantly 1.76 fold lower compared to DF in the febrile phase of illness. Thus, serotonin alone provided good prognostic utility (Area Under Curve, AUC of serotonin = 0.8). Additionally, immune mediators associated with DHF may further increase the predictive ability than just serotonin alone. Nine cytokines, including IFN-γ, IL-1β, IL-4, IL-8, G-CSF, MIP-1β, FGF basic, TNFα and RANTES were significantly different between DF and DHF, among which IFN-γ ranked top by multivariate statistics. Combining serotonin and IFN-γ improved the prognosis performance (AUC = 0.92, sensitivity = 77.8%, specificity = 95.8%), suggesting this duplex panel as accurate metrics for the early prognosis of DHF.

Background: Although international guideline recommended routine intracranial pressure (ICP) monitoring for patients with severe traumatic brain injury(TBI), there were conflicting outcomes attributable to ICP monitoring according to the published studies. Hence, we conducted a meta-analysis to evaluate the efficacy and safety of ICP monitoring in patients with TBI., Methods: Based on previous reviews, PubMed and two Chinese databases (Wangfang and VIP) were further searched to identify eligible studies. The primary outcome was mortality. Secondary outcomes included unfavourable outcome, adverse events, length of ICU stay and length of hospital stay. Weighted mean difference (WMD), odds ratio (OR) and 95% confidence intervals (CIs) were calculated and pooled using fixed-effects or random-effects model., Results: two randomized controlled trials (RCTs) and seven cohort studies involving 11,038 patients met the inclusion criteria. ICP monitoring was not associated with a significant reduction in mortality (OR, 1.16; 95% CI, 0.87-1.54), with substantial heterogeneity (I(2) = 80%, P<0.00001), which was verified by the sensitivity analyses. No significant difference was found in the occurrence of unfavourable outcome (OR, 1.40; 95% CI, 0.99-1.98; I(2) = 4%, P = 0.35) and adverse events (OR, 1.04; 95% CI, 0.64-1.70; I(2) = 78%, P = 0.03). However, we should be cautious to the result of adverse events because of the substantial heterogeneity in the comparison. Furthermore, longer ICU and hospital stay were the consistent tendency according to the pooled studies., Conclusions: No benefit was found in patients with TBI who underwent ICP monitoring. Considering substantial clinical heterogeneity, further large sample size RCTs are needed to confirm the current findings.

Background: Dengue virus (DENV) is the most widespread arbovirus with an estimated 100 million infections occurring every year. Endemic in the tropical and subtropical areas of the world, dengue fever/dengue hemorrhagic fever (DF/DHF) is emerging as a major public health concern. The complex array of concurrent host physiologic changes has hampered a complete understanding of underlying molecular mechanisms of dengue pathogenesis., Methodology/principle Findings: Systems level characterization of serum metabolome and lipidome of adult DF patients at early febrile, defervescence, and convalescent stages of DENV infection was performed using liquid chromatography- and gas chromatography-mass spectrometry. The tractability of following metabolite and lipid changes in a relatively large sample size (n = 44) across three prominent infection stages allowed the identification of critical physiologic changes that coincided with the different stages. Sixty differential metabolites were identified in our metabolomics analysis and the main metabolite classes were free fatty acids, acylcarnitines, phospholipids, and amino acids. Major perturbed metabolic pathways included fatty acid biosynthesis and β-oxidation, phospholipid catabolism, steroid hormone pathway, etc., suggesting the multifactorial nature of human host responses. Analysis of phospholipids and sphingolipids verified the temporal trends and revealed association with lymphocytes and platelets numbers. These metabolites were significantly perturbed during the early stages, and normalized to control levels at convalescent stage, suggesting their potential utility as prognostic markers., Conclusions/significance: DENV infection causes temporally distinct serum metabolome and lipidome changes, and many of the differential metabolites are involved in acute inflammatory responses. Our global analyses revealed early anti-inflammatory responses working in concert to modulate early pro-inflammatory processes, thus preventing the host from development of pathologies by excessive or prolonged inflammation. This study is the first example of how an omic- approach can divulge the extensive, concurrent, and dynamic host responses elicited by DENV and offers plausible physiological insights to why DF is self limiting.

A new porcine parvovirus (PPV), provisionally designated as PPV5, was identified in U.S. pigs. Cloning and sequencing from a circular or head-to-tail concatemeric array revealed that the PPV5 possesses the typical genomic organization of parvoviruses with two major predicted open reading frames (ORF1 and ORF2), and is most closely related to PPV4 with overall genomic identities of 64.1-67.3%. The amino acid identities between PPV5 and PPV4 were 84.6%-85.1% for ORF1 and 54.0%-54.3% for ORF2. Unlike PPV4, but similar to bovine parvovirus 2 (BPV2), PPV5 lacks the additional ORF3 and has a much longer ORF2. Moreover, the amino acid sequences of ORF1 and ORF2 of BPV2 showed higher homologies to PPV5 than to PPV4. The conserved motifs of the Ca(2+) binding loop (YXGXG) and the catalytic center (HDXXY) of phospholipase A2 (PLA2) were identified in VP1 (ORF2) of PPV5, as well as in BPV2, but were not present in PPV4. Phylogenetic analyses revealed that PPV5, PPV4 and BPV2 form a separate clade different from the genera Parvovirus and Bocavirus. Further epidemiologic investigations of PPV4 and PPV5 in U.S. pigs of different ages indicated a slightly higher prevalence for PPV5 (6.6%; 32/483) compared to PPV4 (4.1%; 20/483), with detection of concurrent PPV4 and PPV5 in 15.6% (7/45) of lungs of infected pigs. Evidence for potential vertical transmission or association with reproductive failure was minimal for both PPV4 and PPV5. The high similarity to PPV4 and the lack of ORF3 may suggest PPV5 is an intermediate of PPV4 during the evolution of parvoviruses in pigs.

Since the KCNB1 encoding Kv2.1 channel accounts for the majority of Kv currents modulating insulin secretion by pancreatic islet beta-cells, we postulated that KCNB1 is a plausible candidate gene for genetic variation contributing to the variable compensatory secretory function of beta-cells in type-2 diabetes (T2D). We conducted two studies, a case-control study and a cross-section study, to investigate the association of common single-nucleotide polymorphisms (SNPs) in KCNB1 with T2D and its linking traits. In the case-control study, we first examined the association of 20 tag SNPs of KCNB1 with T2D in a population with 226 T2D patients and non-diabetic subjects (screening study). We then identified the association in an enlarged population of 412 T2D patients and non-diabetic subjects (replication study). In the cross-sectional study, we investigated the linkage between the candidate SNP rs1051295 and T2D by comparing beta-cell function and insulin sensitivity among rs1051295 genotypes in a general population of 1051 subjects at fasting and after glucose loading (oral glucose tolerance tests, OGTT) in 84 fasting glucose impaired subjects, and several T2D-related traits. We found that among the 19 available tag SNPs, only the KCNB1 rs1051295 was associated with T2D (P = 0.027), with the rs1051295 TT genotype associated with an increased risk of T2D compared with genotypes CC (P = 0.009). At fasting, rs1051295 genotype TT was associated with a 9.8% reduction in insulin sensitivity compared to CC (P = 0.008); along with increased plasma triglycerides (TG) levels (TT/CC: P = 0.046) and increased waist/hip (W/H) ratio (TT/CC: P = 0.013; TT/TC: P = 0.002). OGTT confirmed that genotype TT exhibited reduced insulin sensitivity by 16.3% (P = 0.030) compared with genotype TC+CC in a fasting glucose impaired population. The KCNB1 rs1051295 genotype TT in the Chinese Han population is associated with decreased insulin sensitivity and increased plasma TG and W/H ratio, which together contribute to an increased risk for T2D.

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2'-O methyltransferase activities that are required for the formation of 5' type I cap (m(7)GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2'-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2'-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2'-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2'-O-methyladenosine. The 2'-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2'-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2'-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.