Optimized plasmid loading of human erythrocytes for Plasmodium falciparum DNA transfections.

In vitro modification of Plasmodium falciparum genes is the cornerstone of basic and translational malaria research. Achieved through DNA transfection, these modifications may entail altering protein sequence or abundance. Such experiments are critical for defining the molecular mechanisms of key parasite phenotypes and for validation of drug and vaccine targets. Despite its importance, successful transfection remains difficult and is a resource-intensive, rate-limiting step in P. falciparum research. Here, we report that inefficient loading of plasmid into erythrocytes limits transfection efficacy with commonly used electroporation methods. As these methods also require expensive instrumentation and consumables that are not broadly available, we explored a simpler method based on plasmid loading through hypotonic lysis and resealing of erythrocytes. We used parasite expression of a sensitive NanoLuc reporter for rapid evaluation and optimization of each step. Hypotonic buffer composition, resealing buffer volume and composition, and subsequent incubation affected plasmid retention and successful transfection. While ATP was critical for erythrocyte resealing, addition of Ca++ or glutathione did not improve transfection efficiency, with increasing Ca++ concentrations proving detrimental to outcomes. Compared with either the standard electroporation method or a previously reported hypotonic loading protocol, the optimized method yields greater plasmid loading and higher expression of the NanoLuc reporter 48 h after transfection. It also produced significantly faster outgrowth of parasites in transfections utilizing either episomal expression or CRISPR-Cas9 mediated integration. This new method produces higher P. falciparum transfection efficiency, reduces resource requirements and should accelerate molecular studies of malaria drug and vaccine targets.

A case of Adult-onset Acute Flaccid Myelitis Accompanied by Rhombencephalitis which First Presented with Prominent Psychiatric Symptoms and Dysautonomia Mimicking Anti-N-methyl-d-aspartate Receptor Encephalitis.

A 44-year-old woman with a subacute onset of an altered mental status, urinary retention, and fluctuating blood pressure was initially diagnosed with anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis, meeting the criteria of Graus et al. Cardiac arrest occurred, which required pacemaker placement. She subsequently showed profound flaccid limb paralysis, with magnetic resonance imaging demonstrating focal necrotic lesions localized in the anterior horn of the longitudinal segments of the spinal cord and in the pontine tegmentum. Enteroviruses or autoimmune encephalitis-associated autoantibodies were not detected. We herein report a case of acute flaccid myelitis with profound psychiatric symptoms and dysautonomia, resembling NMDAR encephalitis.

Oncogenic K-RasG12V cannot overcome proliferation failure caused by loss of Ppp6c in mouse embryonic fibroblasts.

Protein phosphatase 6 is a Ser/Thr protein phosphatase and its catalytic subunit is Ppp6c. Ppp6c is thought to be indispensable for proper growth of normal cells. On the other hand, loss of Ppp6c accelerates growth of oncogenic Ras-expressing cells. Although it has been studied in multiple contexts, the role(s) of Ppp6c in cell proliferation remains controversial. It is unclear how oncogenic K-Ras overcomes cell proliferation failure induced by Ppp6c deficiency; therefore, in this study, we attempted to shed light on how oncogenic K-Ras modulates tumor cell growth. Contrary to our expectations, loss of Ppp6c decreased proliferation, anchorage-independent growth in soft agar, and tumor formation of oncogenic Ras-expressing mouse embryonic fibroblasts (MEFs). These findings show that oncogenic K-RasG12V cannot overcome proliferation failure caused by loss of Ppp6c in MEFs.

Risk of transfusion-transmitted infection with severe acute respiratory syndrome coronavirus 2 from blood donors in Japan.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) essentially affects respiratory organs and tissues. SARS-CoV-2 RNAemia is often associated with more severe cases of coronavirus disease 2019 (COVID-19) compared to cases without RNAemia. To determine the impact of the pandemic on transfusion medicine, particularly transfusion-related infection, we examined the frequency of blood donation with RNAemia, the viral RNA (vRNA) concentration, and any possibility of transfusion-transmitted infection (TTI) among transfusion recipients. STUDY DESIGN AND METHODS: vRNA was examined in plasma/serum samples from 496 of 513 blood donors who reported having been infected with SARS-CoV-2 within 2 weeks of donation among a total of ca. 9.9 million blood donations in Japan between January 15, 2020, and December 31, 2021. The clinical course of patients transfused with the blood component containing vRNA was also examined. RESULTS: vRNA was detected in 23 of 496 samples. The median period from blood donation to COVID-19 onset was 1 day in 16 RNAemia-positive donors. Most samples had vRNA concentrations below the limit of quantification. Three patients were transfused with either a packed red blood cell or platelet concentrate that tested positive for vRNA, showing no COVID-19 symptoms and testing negative for vRNA in post-transfusion blood. CONCLUSION: The rate of RNAemia was 4.6% among blood donors who were found to be infected with SARS-CoV-2 shortly after donation, and vRNA concentrations in their donated blood were extremely low. There was no evidence of TTI in the recipients transfused with RNAemia-positive blood components. TTI risk in SARS-CoV-2 is negligible.

Optimized Pyridazinone Nutrient Channel Inhibitors Are Potent and Specific Antimalarial Leads.

Human and animal malaria parasites increase their host erythrocyte permeability to a broad range of solutes as mediated by parasite-associated ion channels. Molecular and pharmacological studies have implicated an essential role in parasite nutrient acquisition, but inhibitors suitable for development of antimalarial drugs are missing. Here, we generated a potent and specific drug lead using Plasmodium falciparum, a virulent human pathogen, and derivatives of MBX-2366, a nanomolar affinity pyridazinone inhibitor from a high-throughput screen. As this screening hit lacks the bioavailability and stability needed for in vivo efficacy, we synthesized 315 derivatives to optimize drug-like properties, establish target specificity, and retain potent activity against the parasite-induced permeability. Using a robust, iterative pipeline, we generated MBX-4055, a derivative active against divergent human parasite strains. MBX-4055 has improved oral absorption with acceptable in vivo tolerability and pharmacokinetics. It also has no activity against a battery of 35 human channels and receptors and is refractory to acquired resistance during extended in vitro selection. Single-molecule and single-cell patch-clamp indicate direct action on the plasmodial surface anion channel, a channel linked to parasite-encoded RhopH proteins. These studies identify pyridazinones as novel and tractable antimalarial scaffolds with a defined mechanism of action. SIGNIFICANCE STATEMENT: Because antimalarial drugs are prone to evolving resistance in the virulent human P. falciparum pathogen, new therapies are needed. This study has now developed a novel drug-like series of pyridazinones that target an unexploited parasite anion channel on the host cell surface, display excellent in vitro and in vivo ADME properties, are refractory to acquired resistance, and demonstrate a well defined mechanism of action.

Analysis of N- and O-Linked Glycosylation: Differential Glycosylation after Rat Spinal Cord Injury.

Glycosylation is a fundamental cellular process that has a dramatic impact on the functionality of glycoconjugates such as proteins or lipids and mediates many different biological interactions including cell migration, cellular signaling, and synaptic interactions in the nervous system. In spinal cord injury (SCI), all of these cellular processes are altered, but the potential contributions of glycosylation changes to these alterations has not been thoroughly investigated. We studied the glycosylation of injured spinal cord tissue from rats that received a contusion SCI. The N- and O-linked glycosylation was assessed at 3 and 14 days post-injury (DPI), and compared with uninjured control and time-matched sham spinal tissue. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS/MS) were performed to analyze carbohydrate structures. Results revealed diverse and abundant glycosylation in all groups, with some carbohydrate structures differentially produced in SCI animals compared with uninjured controls and shams. One such change occurred in the abundance of the Sda structure, Neu5Ac-α-(2,3)-[GalNAc-ß-(1,4)-]Gal-ß-(1,4)-GlcNAc, which was increased in SCI samples compared with shams and non-injured controls. Immunohistochemistry (IHC) and western blot were performed on SCI and sham samples using the CT1 antibody, which recognizes the terminal trisaccharide of Sda with high specificity. Both of these metrics confirmed elevated Sda structure in SCI tissue, where IHC further showed that Sda is expressed mainly by microglia. The results of these studies suggest that SCI causes a significant alteration in N- and O-linked glycosylation.

Massive obstetric hemorrhage during cesarean section in a patient after conception by frozen-thawed embryo transfer: a case report.

BACKGROUND: Placenta accreta is a major cause of massive obstetric hemorrhage during cesarean section. In recent years, pregnancy by in vitro fertilization-embryo transfer has been reported as a risk factor for placenta accreta. CASE PRESENTATION: A 36-year-old G1P0 woman with systemic lupus erythematosus became pregnant by frozen-thawed embryo transfer. Emergency cesarean section was performed under general anesthesia due to the diagnosis of non-reassuring fetal status. The placenta invaded the myometrium and completely covered the entire anterior uterine wall. Following birth, 3000 mL of blood loss required rapid fluid infusion and blood transfusion. Total hysterectomy was performed because the placenta could not be separated from the uterine wall. Histological examination revealed placenta accreta/increta. CONCLUSIONS: When performing cesarean section on patients who have undergone frozen-thawed embryo transfer, preoperative examinations to assess for placenta accreta should be performed, and the anesthetic management should include sufficient planning for massive obstetric hemorrhage.

Long noncoding RNA UCA1 enhances sensitivity to oncolytic vaccinia virus by sponging miR-18a/miR-182 and modulating the Cdc42/filopodia axis in colorectal cancer.

The promising anti-tumor effects of oncolytic vaccinia virus (OVV) have been demonstrated. Further, we previously showed that long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) enhances OVV cell-to-cell spread via the activation of Cdc42 in ovarian cancer. However, its role in other cancer types and the molecular mechanism underlying its effects remain to be explored. In this study, we first demonstrated that UCA1 upregulates OVV cell-to-cell spread but not its binding, entry, and replication in colorectal cancer cells. Functional analysis indicated that Cdc42 activation and filopodia formation play an important role in this process. Moreover, expression analysis of various miRNAs suggested that UCA1 inhibits both miR-18a and miR-182, thereby promoting Cdc42 activation, which in turn, regulates OVV cell-to-cell spread. Furthermore, UCA1 was found to modulate tumor malignancy, drug resistance, and sensitivity to OVV via different miRNAs in colorectal cancer. These findings indicate that a three-marker panel, which includes UCA1 expression, Cdc42 activation, and filopodia formation, could potentially be used to predict the therapeutic effect of OVV in colorectal cancer.

Partial Deletion of Glycoprotein B5R Enhances Vaccinia Virus Neutralization Escape while Preserving Oncolytic Function.

Vaccinia virus (VV) has been utilized in oncolytic virotherapy, but it risks a host antiviral immune response. VV has an extracellular enveloped virus (EEV) form consisting of a normal virion covered with a host-derived outer membrane that enables its spread via circulation while evading host immune mechanisms. However, the immune resistance of EEV is only partial, owing to expression of the surface protein B5R, which has four short consensus repeat (SCR) domains that are targeted by host immune factors. To engineer a more effective virus for oncolytic virotherapy, we developed an enhanced immune-evading oncolytic VV by removing the SCRs from the attenuated strain LC16mO. Although deletion of only the SCRs preserved viral replication, progeny production, and oncolytic activity, deletion of whole B5R led to attenuation of the virus. Importantly, SCR-deleted EEV had higher neutralization resistance than did B5R-wild-type EEV against VV-immunized animal serum; moreover, it retained oncolytic function, thereby prolonging the survival of tumor-bearing mice treated with anti-VV antibody. These results demonstrate that partial SCR deletion increases neutralization escape without affecting the oncolytic potency of VV, making it useful for the treatment of tumors under the anti-virus antibody existence.

lncRNA UCA1-Mediated Cdc42 Signaling Promotes Oncolytic Vaccinia Virus Cell-to-Cell Spread in Ovarian Cancer.

Oncolytic vaccinia virus (OVV) has demonstrated appropriate safety profiles for clinical development. Although designed to kill cancer cells efficiently, OVV sensitivity varies in individual cancers, and predictive biomarkers of therapeutic responses have not been identified. Here we found that OVV was much more efficient in KFTX paclitaxel-resistant ovarian cancer cells compared to that in KFlow paclitaxel-sensitive cells. Microarray analysis identified long non-coding RNA urothelial carcinoma-associated 1 (UCA1) upregulation, which contributed to both enhanced paclitaxel resistance and OVV spread. In addition, UCA1 expression correlated with efficient OVV spread in other ovarian cell lines and primary cancer cell cultures. When host pathways underlying OVV spread were analyzed, differences were detected in the activation of the Rho GTPase Cdc42, suggesting that filopodia formation enhances OVV cell-to-cell spread and tumor migration. Moreover, we established a clinically relevant mouse model of peritoneal metastasis using KFTX or KFlow cells. Paclitaxel exerted anti-tumor effects on KFlow, but not KFTX, tumors. In mice bearing KFTX cells after paclitaxel failure, OVV treatment induced the regression of residual tumors and improved survival. Our findings demonstrated that UCA1 promotes OVV cell-to-cell spread in ovarian cancer, resulting in enhanced therapeutic outcome.

Relapse Prevention in Ulcerative Colitis by Plant-Based Diet Through Educational Hospitalization: A Single-Group Trial.

CONTEXT: No known published study has focused on a plant-based diet (PBD) in the treatment of ulcerative colitis (UC). OBJECTIVE: To investigate relapse prevention in UC after consumption of a PBD during educational hospitalization in Japan. DESIGN: Prospective study of patients with mild UC or UC in remission who did not need immediate treatment. A PBD and dietary guidance were provided during a two-week hospitalization. MAIN OUTCOME MEASURES: The primary end point was relapse (a flare-up that required more aggressive treatment) during the follow-up period. Kaplan-Meier analysis was used to calculate the cumulative relapse rate. Secondary end points were immediate improvement in symptoms or laboratory data during hospitalization and a chronologic change in the PBD score, which evaluated adherence to the PBD. RESULTS: Sixty cases were studied: 29 initial episode cases and 31 relapse cases. Of these, 31 involved proctitis; 7, left-sided colitis; and 22, extensive colitis. Thirty-seven patients were receiving medication; 23 were not. The median age was 34 years; median follow-up was 3 years 6 months. Eight cases relapsed during follow-up. The cumulative relapse rates at 1, 2, 3, 4, and 5 years of follow-up were 2%, 4%, 7%, 19%, and 19%, respectively. Most patients (77%) experienced some improvement such as disappearance or decrease of bloody stool during hospitalization. The short- and long-term PBD scores after the hospitalization were higher than baseline PBD scores. CONCLUSION: Relapse rates after educational hospitalization providing a PBD were far lower than those reported with medication. Educational hospitalization is effective at inducing habitual dietary changes.

Induction with Infliximab and a Plant-Based Diet as First-Line (IPF) Therapy for Crohn Disease: A Single-Group Trial.

BACKGROUND: Approximately 30% of patients with Crohn disease (CD) are unresponsive to biologics. No previous study has focused on a plant-based diet in an induction phase of CD treatment. OBJECTIVE: To investigate the remission rate of infliximab combined with a plant-based diet as first-line (IPF) therapy for CD. METHODS: This was a prospective single-group trial conducted at tertiary hospitals. Subjects included consecutive adults with a new diagnosis (n = 26), children with a new diagnosis (n = 11), and relapsing adults (n = 9) with CD who were naïve to treatment with biologics. Patients were admitted and administered a standard induction therapy with infliximab (5 mg/kg; 3 infusions at 0, 2, and 6 weeks). Additionally, they received a lacto-ovo-semivegetarian diet. The primary end point was remission, defined as the disappearance of active CD symptoms at week 6. Secondary end points were Crohn Disease Activity Index (CDAI) score, C-reactive protein (CRP) concentration, and mucosal healing. RESULTS: Two adults with a new diagnosis were withdrawn from the treatment protocol because of intestinal obstruction. The remission rates by the intention-to-treat and per-protocol analyses were 96% (44/46) and 100% (44/44), respectively. Mean CDAI score (314) on admission decreased to 63 at week 6 (p < 0.0001). Mean CRP level on admission (5.3 mg/dL) decreased to 0.2 (p < 0.0001). Mucosal healing was achieved in 46% (19/41) of cases. CONCLUSION: IPF therapy can induce remission in most patients with CD who are naïve to biologics regardless of age or whether they have a new diagnosis or relapse.

Stereoselective Synthesis of Nitrogen-Containing Compounds from Enamines.

The domino reaction of enamines, electrophiles (N-sulfonylimines, N-tosylisocyanate, or diethyl azodicarboxylate), and trichlorosilane provided trans-amines (trans/cis = > 99:1 to 96:4). Meanwhile, the sequential imino ene-type reaction of enamines and electrophiles/NaBH3CN reduction afforded cis-amines (trans/cis = 1:>99 to 15:85). The reversal of selectivity is discussed on the basis of diastereofacial selection of the plausible iminium ion intermediates. For the domino reaction of cyclic enamines and cyclic imines, high enantioselectivity (er = 95.7:4.3 to 99.9:0.1) was achieved by utilizing chiral Lewis base catalysts.

Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

AIM: The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. METHODS: Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. RESULTS: Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. CONCLUSION: Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement.

MicroRNAs miR-199a-5p and -3p target the Brm subunit of SWI/SNF to generate a double-negative feedback loop in a variety of human cancers.

The chromatin remodeling complex SWI/SNF is an important epigenetic regulator that includes one Brm or BRG1 molecule as catalytic subunit. Brm and BRG1 do not function identically, so this complex can regulate gene expression either positively or negatively, depending on the promoter to which it is recruited. Notably, Brm attenuation due to posttranscription suppression occurs often in human tumor cells, in which this event contributes to their oncogenic potential. Here, we report that the 3'-untranslated region of Brm mRNA has two sites that are efficiently targeted by the microRNAs miR-199a-5p and -3p, revealing a novel mechanism for modulation of Brm-type SWI/SNF activity. Computational mapping of the putative promoter region of miR-199a-2 (miPPR-199a-2) has defined it as the major contributing genetic locus for miR-199a-5p and-3p production in these tumor cell lines. We validated this predicted region by direct promoter analysis to confirm that Egr1 is a strong positive regulator of the miR-199a-2 gene. Importantly, we also showed that Egr1, miR-199a-5p, and miR-199a-3p are expressed at high levels in Brm-deficient tumor cell lines but only marginally in Brm-expressing tumor cells. Finally, we also obtained evidence that Brm negatively regulates Egr1. Together, our results reveal that miR-199a and Brm form a double-negative feedback loop through Egr1, leading to the generation of these two distinct cell types during carcinogenesis. This mechanism may offer a partial explanation for why miR-199a-5p and -3p have been reported to be either upregulated or downregulated in a variety of tumors.

Coffee consumption enhances high-density lipoprotein-mediated cholesterol efflux in macrophages.

RATIONALE: Association of habitual coffee consumption with coronary heart disease morbidity and mortality has not been established. We hypothesized that coffee may enhance reverse cholesterol transport (RCT) as the antiatherogenic properties of high-density lipoprotein (HDL). OBJECTIVE: This study was to investigate whether the phenolic acids of coffee and coffee regulates RCT from macrophages in vitro, ex vivo and in vivo. METHODS AND RESULTS: Caffeic acid and ferulic acid, the major phenolic acids of coffee, enhanced cholesterol efflux from THP-1 macrophages mediated by HDL, but not apoA-I. Furthermore, these phenolic acids increased both the mRNA and protein levels of ATP-binding cassette transporter (ABC)G1 and scavenger receptor class B type I (SR-BI), but not ABCA1. Eight healthy volunteers were recruited for the ex vivo study, and blood samples were taken before and 30 minutes after consumption of coffee or water in a crossover study. The mRNA as well as protein levels of ABCG1, SR-BI, and cholesterol efflux by HDL were increased in the macrophages differentiated under autologous sera obtained after coffee consumption compared to baseline sera. Finally, effects of coffee and phenolic acid on in vivo RCT were assessed by intraperitoneally injecting [(3)H]cholesterol-labeled acetyl low-density lipoprotein-loaded RAW264.7 cells into mice, then monitoring appearance of (3)H tracer in plasma, liver, and feces. Supporting in vitro and ex vivo data, ferulic acid was found to significantly increase the levels of (3)H tracer in feces. CONCLUSIONS: Coffee intake might have an antiatherogenic property by increasing ABCG1 and SR-BI expression and enhancing HDL-mediated cholesterol efflux from the macrophages via its plasma phenolic acids.

Overexpression of thymosin beta4 increases pseudopodia formation in LNCaP prostate cancer cells.

Thymosin beta4, a major G-actin-sequestering protein, is known to be involved in tumor metastasis. In the present study, we found that thymosin beta4 expression promotes the formation of actin-based pseudopodia-like extensions, associated with cell migration, in human prostate cancer LNCaP cells. Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin and Cdc42/Rac1/RhoA inhibitor Clostridium difficile toxin B significantly reduced pseudopodia formation in thymosin beta4-overexpressing LNCaP cells, suggesting that the pseudopodia formation by thymosin beta4 is probably involved in PI3K and Rho family pathway. We recently reported that thymosin beta4 expression is upregulated by androgen deprivation in prostate cancer cells. The increase in thymosin beta4 may be one of the causes of prostate cancer progression after androgen ablation therapy.

Versatile synthesis of head group functionalized phospholipids via oxime bond formation.

A method for introduction of various head groups on phospholipid frameworks via oxime bond formation has been developed for the synthesis of cyclen-Cu(II), pyrene, naphthalene, and other headgroup functionalized phospholipids that can cleave the membrane protein, hemagglutinin.

Downregulation of thymosin beta4 expression by androgen in prostate cancer LNCaP cells.

Androgen ablation therapy is an effective treatment for advanced prostate cancer, but the tumor often progresses toward a more aggressive phenotype. We determined the changes in genes associated with the malignant progression and found increased thymosin beta4, involved in tumor metastasis, in androgen-sensitive LNCaP cells grown in the medium with androgen-deficient, charcoal-stripped fetal calf serum. The mRNA expression of thymosin beta4 was determined by real-time polymerase chain reaction analysis. The transcriptional activity of thymosin beta4 was measured by luciferase assay using reporter plasmid containing 5'-flanking region of thymosin beta4. Thymosin beta4 mRNA expression was increased in LNCaP cells in the androgen-deficient condition and decreased by dihydrotestosterone treatment. Androgen receptor antagonist bicalutamide inhibited thymosin beta4 expression in a dose-dependent manner. In androgen receptor-negative PC-3 cells, no significant effects on thymosin beta4 gene expression were observed. The regulation of thymosin beta4 mRNA expression by androgen is due to the transcriptional activation. Deletion analysis revealed that the region between -83 bp and -46 bp of the thymosin beta4 gene is responsible for the regulation of the transcriptional activity by androgen. Thymosin beta4 expression is negatively controlled at the transcriptional level by androgen.