Extraction of high quality and high yield RNA from frozen EDTA blood.

Peripheral blood RNA profiling, which can reveal systemic changes in gene expression and immune responses to disease onset and progression, is a powerful tool for diagnosis and biomarker discovery. This technique usually requires high quality RNA, which is only obtainable from fresh blood, or frozen blood that has been collected in special RNA-stabilisation systems. The current study aimed to develop a novel protocol to extract high quality RNA from frozen blood that had been collected in the conventional EDTA tubes. We determined that thawing EDTA blood in the presence of cell lysis/RNA stabilisation buffers (Paxgene or Nucleospin) significantly improved RNA quality (RIN) from below 5 to above 7, which to date has not been shown possible. The EDTA-Nucleospin protocol resulted in 5 times higher yield than the EDTA-Paxgene-PreAnalytix method. The average RIN and mRNA expression levels of five different genes including 18 s, ACTB, MCP1, TNFa and TXNIP using this protocol were also indifferent to those from Paxgene blood, suggesting similar RNA quality and blood transcriptome. Moreover, the protocol allows DNA to be extracted simultaneously. In conclusion, we have developed a practical and efficient protocol to extract high quality, high yield RNA from frozen EDTA blood.

Catatonia and Opioid Withdrawal: A Case Report.

In this case report, we present an 82-year-old female who was diagnosed with catatonia after she exhibited immobility, mutism, withdrawal, and stereotypy during a hospitalization for altered mental status. Fentanyl was found in her urine toxicology, and it was later discovered that she had been taking non-prescription pills from Mexico that were likely the source of the fentanyl. Her catatonia quickly remitted with benzodiazepine treatment. This case underscores previously unknown risks of substance use, which has grown especially important to psychiatric care considering how rampant the opioid epidemic has become. More so, these risks extend beyond opioid use disorders since other non-prescription drugs are commonly laced with fentanyl. Not only does this education need to be given to providers and patients alike, but further research should be conducted to establish and quantify the risk of catatonia with opioid withdrawal.

Lack of SPNS1 results in accumulation of lysolipids and lysosomal storage disease in mouse models.

Accumulation of sphingolipids, especially sphingosines, in the lysosomes is a key driver of several lysosomal storage diseases. The transport mechanism for sphingolipids from the lysosome remains unclear. Here, we identified SPNS1, which shares the highest homology to SPNS2, a sphingosine-1-phosphate (S1P) transporter, functions as a transporter for lysolipids from the lysosome. We generated Spns1-KO cells and mice and employed lipidomic and metabolomic approaches to reveal SPNS1 ligand identity. Global KO of Spns1 caused embryonic lethality between E12.5 and E13.5 and an accumulation of sphingosine, lysophosphatidylcholines (LPC), and lysophosphatidylethanolamines (LPE) in the fetal livers. Similarly, metabolomic analysis of livers from postnatal Spns1-KO mice presented an accumulation of sphingosines and lysoglycerophospholipids including LPC and LPE. Subsequently, biochemical assays showed that SPNS1 is required for LPC and sphingosine release from lysosomes. The accumulation of these lysolipids in the lysosomes of Spns1-KO mice affected liver functions and altered the PI3K/AKT signaling pathway. Furthermore, we identified 3 human siblings with a homozygous variant in the SPNS1 gene. These patients suffer from developmental delay, neurological impairment, intellectual disability, and cerebellar hypoplasia. These results reveal a critical role of SPNS1 as a promiscuous lysolipid transporter in the lysosomes and link its physiological functions with lysosomal storage diseases.

Automated antibody dispensing to improve high-parameter flow cytometry throughput and analysis.

Over the past decade, the flow cytometry field has witnessed significant advancements in the number of fluorochromes that can be detected. This enables researchers to analyze more than 40 markers simultaneously on thousands of cells per second. However, with this increased complexity and multiplicity of markers, the manual dispensing of antibodies for flow cytometry experiments has become laborious, time-consuming, and prone to errors. An automated antibody dispensing system could provide a potential solution by enhancing the efficiency, and by improving data quality by faithfully dispensing the fluorochrome-conjugated antibodies and by enabling the easy addition of extra controls. In this study, a comprehensive comparison of different liquid handlers for dispensing fluorochrome-labeled antibodies was conducted for the preparation of flow cytometry stainings. The evaluation focused on key criteria including dispensing time, dead volume, and reliability of dispensing. After benchmarking, the I.DOT, a non-contact liquid handler, was selected and optimized in more detail. In the end, the I.DOT was able to prepare a 25-marker panel in 20 min, including the full stain, all FMOs and all single stain controls for cells and beads. Having all these controls improved the validation of the panel, visualization, and analysis of the data. Thus, automated antibody dispensing by dispensers such as the I.DOT reduces time and errors, enhances data quality, and can be easily integrated in an automated workflow to prepare samples for flow cytometry.

Preferences for childbirth delivery and pain relief methods among pregnant women in Vietnam.

Background: Understanding childbirth delivery and pain relief method preferences is important as a part of the shared decision-making process between pregnant women and health professionals. This study aimed to examine the preferences for childbirth delivery modes and pain relief methods and factors related to these preferences among pregnant women in Vietnam. Methods: A cross-sectional survey on pregnant women was conducted in two obstetrics hospitals in Vietnam. Face-to-face interviews using a structured questionnaire were performed to collect information about sociodemographic characteristics, pregnancy characteristics, preferences for different childbirth delivery modes, and pain relief methods. Multivariate logistic regression was employed for determining associated factors with the preferences. Results: Of 576 pregnant women, 34% of participants preferred cesarean section. Most of the sample did not have any preferences for specific pharmacological pain relief methods (70.1%), while support from partner/relatives was the most preferable non-pharmacological method (61.3%), following by water birth (11.1%) and acupuncture (9.9%). Desire to have another baby, relatives' experience, selection date of birth, and instrumental social support were major drivers of the cesarean section selection. This preference was an important factor in the preference for pharmacological pain relief. Meanwhile, high levels of informational and emotional support were associated with non-pharmacological method preference. Conclusion: This study highlighted a high preference rate for cesarean section in urban pregnant women in Vietnam. Holistic approaches from family, health facility, and policy should be performed to diminish the cesarean rate preference and promote the use of non-pharmacological pain relief methods during birth.

Effects of pericardiocentesis on renal function and cardiac hemodynamics.

BACKGROUND: Previous case studies have reported reversal of acute renal failure after pericardiocentesis in pericardial effusion. This study examines the effects of pericardiocentesis on preprocedural low cardiac output and acute renal dysfunction in patients with pericardial effusion. METHODS: This is a retrospective study of 95 patients undergoing pericardiocentesis between 2015 and 2020. Pre- and post-procedure transthoracic echocardiograms (TTE) were reviewed for evidence of cardiac tamponade, resolution of pericardial effusion, and for estimation of right atrial (RA) pressure and cardiac output. Laboratory values were compared at presentation and post-procedure. Patients on active renal replacement therapy were excluded. RESULTS: Ninety-five patients were included for analysis (mean age 62.2 ± 17.8 years, 58% male). There was a significant increase in glomerular filtration rate pre- and post-procedure. Fifty-six patients (58.9%) had an improvement in glomerular filtration rate after pericardiocentesis (termed "responders"), and these patients had a lower pre-procedure glomerular filtration rate than "non-responders." There was a significant improvement in estimated cardiac output and right atrial pressure for patients in both groups. Patients who had an improvement in renal function had significantly lower pre-procedural diastolic blood pressure and mean arterial pressure. CONCLUSIONS: Pericardial drainage may improve effusion-mediated acute renal dysfunction by reducing right atrial pressure and thus systemic venous congestion, and by increasing forward stroke volume and perfusion pressure.

Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs.

There is a broad diversity among Cas12a endonucleases that possess nucleic acid detection and gene-editing capabilities, but few are studied extensively. Here, we present an exhaustive investigation of 23 Cas12a orthologs, with a focus on their cis- and trans-cleavage activities in combination with noncanonical crRNAs. Through biochemical assays, we observe that some noncanonical crRNA:Cas12a effector complexes outperform their corresponding wild-type crRNA:Cas12a. Cas12a can recruit crRNA with modifications such as loop extensions and split scaffolds. Moreover, the tolerance of Cas12a to noncanonical crRNA is also observed in mammalian cells through the formation of indels. We apply the adaptability of Cas12a:crRNA complexes to detect SARS-CoV-2 in clinical nasopharyngeal swabs, saliva samples, and tracheal aspirates. Our findings further expand the toolbox for next-generation CRISPR-based diagnostics and gene editing.

Injecting drug use and hepatitis C virus infection independently increase biomarkers of inflammatory disease risk which are incompletely restored by curative direct-acting antiviral therapy.

Background: Hepatitis C virus (HCV) infections are more prevalent in people who inject drugs (PWID) who often experience additional health risks. HCV induces inflammation and immune alterations that contribute to hepatic and non-hepatic morbidities. It remains unclear whether curative direct acting antiviral (DAA) therapy completely reverses immune alterations in PWID. Methods: Plasma biomarkers of immune activation associated with chronic disease risk were measured in HCV-seronegative (n=24) and HCV RNA+ (n=32) PWID at baseline and longitudinally after DAA therapy. Adjusted generalised estimating equations were used to assess longitudinal changes in biomarker levels. Comparisons between community controls (n=29) and HCV-seronegative PWID were made using adjusted multiple regression modelling. Results: HCV-seronegative PWID exhibited significantly increased levels of inflammatory biomarkers including soluble (s) TNF-RII, IL-6, sCD14 and sCD163 and the diabetes index HbA1c as compared to community controls. CXCL10, sTNF-RII, vascular cell adhesion molecule-1 and lipopolysaccharide binding protein (LBP) were additionally elevated in PWID with viremic HCV infection as compared to HCV- PWID. Whilst curative DAA therapy reversed some biomarkers, others including LBP and sTNF-RII remained elevated 48 weeks after HCV cure. Conclusion: Elevated levels of inflammatory and chronic disease biomarkers in PWID suggest an increased risk of chronic morbidities such as diabetes and cardiovascular disease. HCV infection in PWID poses an additional disease burden, amplified by the incomplete reversal of immune dysfunction following DAA therapy. These findings highlight the need for heightened clinical surveillance of PWID for chronic inflammatory diseases, particularly those with a history of HCV infection.

MFSD7c functions as a transporter of choline at the blood-brain barrier.

Mutations in the orphan transporter MFSD7c (also known as Flvcr2), are linked to Fowler syndrome. Here, we used Mfsd7c knockout (Mfsd7c-/-) mice and cell-based assays to reveal that MFSD7c is a choline transporter at the blood-brain barrier (BBB). We performed comprehensive metabolomics analysis and detected differential changes of metabolites in the brains and livers of Mfsd7c-/-embryos. Particularly, we found that choline-related metabolites were altered in the brains but not in the livers of Mfsd7c-/- embryos. Thus, we hypothesized that MFSD7c regulates the level of choline in the brain. Indeed, expression of human MFSD7c in cells significantly increased choline uptake. Interestingly, we showed that choline uptake by MFSD7c is greatly increased by choline-metabolizing enzymes, leading us to demonstrate that MFSD7c is a facilitative transporter of choline. Furthermore, single-cell patch clamp analysis showed that the import of choline by MFSD7c is electrogenic. Choline transport function of MFSD7c was shown to be conserved in vertebrates, but not in yeasts. We demonstrated that human MFSD7c is a functional ortholog of HNM1, the yeast choline importer. We also showed that several missense mutations identified in patients exhibiting Fowler syndrome had abolished or reduced choline transport activity. Mice lacking Mfsd7c in endothelial cells of the central nervous system suppressed the import of exogenous choline from blood but unexpectedly had increased choline levels in the brain. Stable-isotope tracing study revealed that MFSD7c was required for exporting choline derived from lysophosphatidylcholine in the brain. Collectively, our work identifies MFSD7c as a choline exporter at the BBB and provides a foundation for future work to reveal the disease mechanisms of Fowler syndrome.

Sargramostim for Prophylactic Management of Gastrointestinal Immune-Related Adverse Events of Immune Checkpoint Inhibitor Therapy for Cancer.

Immune checkpoint inhibitor (ICI) therapy improves outcomes in several cancers. Unfortunately, many patients experience grade 3-4 treatment-related adverse events, including gastrointestinal (GI) toxicities which are common. These GI immune-related adverse events (irAEs) induced by ICIs present significant clinical challenges, require prompt intervention, and result in treatment delays or discontinuations. The treatment for these potentially severe and even fatal GI irAEs which include enterocolitis, severe diarrhea, and hepatitis may interfere with the anti-cancer approach. Sargramostim (glycosylated, yeast-derived, recombinant human GM-CSF) is an agent that has been used in clinical practice for more than 30 years with a well-recognized safety profile and has been studied in many therapeutic areas. The mechanism of action of sargramostim may treat moderate-to-severe GI irAEs without impairing the anti-cancer therapy. Some early data also suggest a potential survival benefit. Through the differentiation/maturation of monocytes, macrophages, and neutrophils and induction of anti-inflammatory T cell responses, GM-CSF aids in GI homeostasis, mucosal healing, and mucosal immunity. GM-CSF knockout mice are susceptible to severe colitis which was prevented with murine GM-CSF administration. For some patients with GI mucosa and immune cell function impairment, e.g., Crohn's disease, sargramostim reduces disease severity. In a prospective, randomized study (ECOG 1608), advanced melanoma patients had a reduction in grade 3-5 GI irAEs and less frequent colonic perforation in the sargramostim plus ipilimumab arm compared to ipilimumab alone. Sargramostim continues to be studied with ICIs for the prophylactic management of irAEs while also potentially providing a survival benefit.

SARS-CoV-2 infection elucidates unique features of pregnancy-specific immunity.

Pregnancy is a risk factor for increased severity of SARS-CoV-2 and other respiratory infections. The mechanisms underlying this risk have not been well-established, partly due to a limited understanding of how pregnancy shapes immune responses. To gain insight into the role of pregnancy in modulating immune responses at steady state and upon perturbation, we collected peripheral blood mononuclear cells (PBMC), plasma, and stool from 226 women, including 152 pregnant individuals (n = 96 with SARS-CoV-2 infection and n = 56 healthy controls) and 74 non-pregnant women (n = 55 with SARS-CoV-2 and n = 19 healthy controls). We found that SARS-CoV-2 infection was associated with altered T cell responses in pregnant compared to non-pregnant women. Differences included a lower percentage of memory T cells, a distinct clonal expansion of CD4-expressing CD8 + T cells, and the enhanced expression of T cell exhaustion markers, such as programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain-3 (Tim-3), in pregnant women. We identified additional evidence of immune dysfunction in severely and critically ill pregnant women, including a lack of expected elevation in regulatory T cell (Treg) levels, diminished interferon responses, and profound suppression of monocyte function. Consistent with earlier data, we found maternal obesity was also associated with altered immune responses to SARS-CoV-2 infection, including enhanced production of inflammatory cytokines by T cells. Certain gut bacterial species were altered in pregnancy and upon SARS-CoV-2 infection in pregnant individuals compared to non-pregnant women. Shifts in cytokine and chemokine levels were also identified in the sera of pregnant individuals, most notably a robust increase of interleukin-27 (IL-27), a cytokine known to drive T cell exhaustion, in the pregnant uninfected control group compared to all non-pregnant groups. IL-27 levels were also significantly higher in uninfected pregnant controls compared to pregnant SARS-CoV-2-infected individuals. Using two different preclinical mouse models of inflammation-induced fetal demise and respiratory influenza viral infection, we found that enhanced IL-27 protects developing fetuses from maternal inflammation but renders adult female mice vulnerable to viral infection. These combined findings from human and murine studies reveal nuanced pregnancy-associated immune responses, suggesting mechanisms underlying the increased susceptibility of pregnant individuals to viral respiratory infections.

Cellulose nanocrystals extracted from rice husk using the formic/peroxyformic acid process: isolation and structural characterization.

Cellulose derived from biomass is a renewable resource with numerous applications. Using formic/peroxyformic acid at atmospheric pressure, cellulose nanocrystals (CNC) were isolated from rice husk (RH) in this study. This method was an excellent way to get rid of lignin and hemicelluloses from RH. The cellulose was subsequently acid hydrolyzed by H2SO4 (64%) for 30 minutes at 45 °C. The chemical and microstructure analysis showed that the lignin and hemicellulose contents of raw RH had been eliminated, and the crystallinity content of CNC was 67.16%. According to transmission electron microscopy (TEM) morphological analysis, CNC measured 19 ± 3.3 nm in diameter, 195 ± 24 nm in length, and 10.2 ± 6.8 in aspect ratio. The thermal stability of RH and CNC was also investigated using thermogravimetric analysis (TGA). These encouraging findings demonstrated the potential for reusing RH agricultural waste to create CNC and include nanocomposites as a reinforcing material.

Priority populations' experiences of isolation, quarantine and distancing for COVID-19: protocol for a longitudinal cohort study (Optimise Study).

INTRODUCTION: Longitudinal studies can provide timely and accurate information to evaluate and inform COVID-19 control and mitigation strategies and future pandemic preparedness. The Optimise Study is a multidisciplinary research platform established in the Australian state of Victoria in September 2020 to collect epidemiological, social, psychological and behavioural data from priority populations. It aims to understand changing public attitudes, behaviours and experiences of COVID-19 and inform epidemic modelling and support responsive government policy. METHODS AND ANALYSIS: This protocol paper describes the data collection procedures for the Optimise Study, an ongoing longitudinal cohort of ~1000 Victorian adults and their social networks. Participants are recruited using snowball sampling with a set of seeds and two waves of snowball recruitment. Seeds are purposively selected from priority groups, including recent COVID-19 cases and close contacts and people at heightened risk of infection and/or adverse outcomes of COVID-19 infection and/or public health measures. Participants complete a schedule of monthly quantitative surveys and daily diaries for up to 24 months, plus additional surveys annually for up to 48 months. Cohort participants are recruited for qualitative interviews at key time points to enable in-depth exploration of people's lived experiences. Separately, community representatives are invited to participate in community engagement groups, which review and interpret research findings to inform policy and practice recommendations. ETHICS AND DISSEMINATION: The Optimise longitudinal cohort and qualitative interviews are approved by the Alfred Hospital Human Research Ethics Committee (# 333/20). The Optimise Study CEG is approved by the La Trobe University Human Ethics Committee (# HEC20532). All participants provide informed verbal consent to enter the cohort, with additional consent provided prior to any of the sub studies. Study findings will be disseminated through public website (https://optimisecovid.com.au/study-findings/) and through peer-reviewed publications. TRIAL REGISTRATION NUMBER: NCT05323799.

Derivation and External Validation of the Ottawa Bloodstream Infection Model for Acutely Ill Adults.

BACKGROUND: Knowing the probability that patients have a bloodstream infection (BSI) could influence the ordering of blood cultures and interpretation of their preliminary results. Many previous BSI probability models have limited applicability and accuracy. This study used currently recommended modeling techniques and a large sample to derive and validate the Ottawa BSI Model. METHODS: At a tertiary care teaching hospital, we retrieved a random sample of 4180 adults having blood cultures in our emergency department or during the initial 48 h of the encounter. Variable selection was based on clinical experience and a systematic review of previous model performance. Model performance was measured in a temporal external validation group of 4680 patients. RESULTS: A total of 327 derivation patients had a BSI (8.0%). BSI risk increased with increased number of culture sets (2 sets: adjusted odds ratio [aOR] 1.52 [1.10-2.11]; 3 sets: 1.99 [0.86-4.58]); with indwelling catheter (aOR 2.07 [1.34-3.20); with increasing temperature, heart rate, and neutrophil-lymphocyte ratio; and with decreasing systolic blood pressure, platelet count, urea-creatinine ratio, and estimated glomerular filtration rate. In the temporal external validation group, model discrimination was good (c-statistic 0.71 [0.69-0.74]) and calibration was very good (integrated calibration index .016 [.010-.024]). Exclusion of validation patients with acute SARS-CoV-2 infection improved discrimination slightly (c-statistic 0.73 [0.69-0.76]). CONCLUSIONS: The Ottawa BSI Model uses commonly available data to return an expected BSI probability for acutely ill patients. However, it cannot exclude BSI and its complexity requires computational assistance to use.

CRISPR-based diagnostics detects invasive insect pests.

Rapid identification of organisms is essential for many biological and medical disciplines, from understanding basic ecosystem processes, disease diagnosis, to the detection of invasive pests. CRISPR-based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR-based diagnostic developed with the universal cytochrome-oxidase 1 gene (CO1). The CO1 gene is the most sequenced gene among Animalia, and therefore our approach can be adopted to detect nearly any animal. We tested the approach on three difficult-to-identify moth species (Keiferia lycopersicella, Phthorimaea absoluta and Scrobipalpa atriplicella) that are major invasive pests globally. We designed an assay that combines recombinase polymerase amplification (RPA) with CRISPR for signal generation. Our approach has a much higher sensitivity than real-time PCR assays and achieved 100% accuracy for identification of all three species, with a detection limit of up to 120 fM for P. absoluta and 400 fM for the other two species. Our approach does not require a sophisticated laboratory, reduces the risk of cross-contamination, and can be completed in less than 1 h. This work serves as a proof of concept that has the potential to revolutionize animal detection and monitoring.

Data on chemical composition of coffee husks and lignin microparticles as their extracted product.

Coffee husks are an abundant and underutilized biomass waste released from coffee production. Experimental analysis showed that coffee husks consisted of 39.2 ± 0.2 wt% cellulose, 12.6 ± 0.1 wt% hemicellulose, 23.3 ± 0.1 wt% Klason lignin, 2.9 ± 0.4 wt% acid-soluble lignin, 8.7 ± 0.2 wt% extractives, and 9.5 ± 0.2 wt% ash. Moreover, different minor elements, including K, Ca, Mg, Al, Fe, Ti, S, and Si, were found. Subsequently, coffee husks were used for the extraction of lignin using an alkaline treatment. As a result, lignin microparticles were formed with a relatively uniform size of 0.55 ± 0.11 mm. Altogether, the current article provided useful data for the valorization of coffee husks and the primary properties of lignin microparticles for further use.

Mfsd7b facilitates choline transport and missense mutations affect choline transport function.

MFSD7b belongs to the Major Facilitator Superfamily of transporters that transport small molecules. Two isoforms of MFSD7b have been identified and they are reported to be heme exporters that play a crucial role in maintaining the cytosolic and mitochondrial heme levels, respectively. Mutations of MFSD7b (also known as FLVCR1) have been linked to retinitis pigmentosa, posterior column ataxia, and hereditary sensory and autonomic neuropathy. Although MFSD7b functions have been linked to heme detoxification by exporting excess heme from erythroid cells, it is ubiquitously expressed with a high level in the kidney, gastrointestinal tract, lungs, liver, and brain. Here, we showed that MFSD7b functions as a facilitative choline transporter. Expression of MFSD7b slightly but significantly increased choline import, while its knockdown reduced choline influx in mammalian cells. The influx of choline transported by MFSD7b is dependent on the expression of choline metabolizing enzymes such as choline kinase (CHKA) and intracellular choline levels, but it is independent of gradient of cations. Additionally, we showed that choline transport function of Mfsd7b is conserved from fly to man. Employing our transport assays, we showed that missense mutations of MFSD7b caused reduced choline transport functions. Our results show that MFSD7b functions as a facilitative choline transporter in mammalian cells.