Title: Brain Plaques Signal Alzheimer's Even Before Other Symptoms Emerge: Study
Category: Health News
Created: 4/13/2020 12:00:00 AM
Last Editorial Review: 4/14/2020 12:00:00 AM

Title: Potato & Sausages, Cold Cuts a Bad Combo for Your Brain
Category: Health News
Created: 4/22/2020 12:00:00 AM
Last Editorial Review: 4/23/2020 12:00:00 AM

Title: Nose Jobs Make Women Look Years Younger, Robot Says
Category: Health News
Created: 1/28/2020 12:00:00 AM
Last Editorial Review: 1/28/2020 12:00:00 AM

Title: Another HIV Hazard: Higher Risk for COPD
Category: Health News
Created: 2/20/2020 12:00:00 AM
Last Editorial Review: 2/21/2020 12:00:00 AM

Title: lopinavir and ritonavir (Kaletra): Potential COVID-19 Drug
Category: Medications
Created: 3/12/2001 12:00:00 AM
Last Editorial Review: 4/8/2020 12:00:00 AM

Title: Money Not a Good Measure of Your Self-Worth
Category: Health News
Created: 4/14/2020 12:00:00 AM
Last Editorial Review: 4/15/2020 12:00:00 AM

Title: Heavy Pot Use Linked to Mental Problems, Even After Quitting
Category: Health News
Created: 4/30/2020 12:00:00 AM
Last Editorial Review: 5/1/2020 12:00:00 AM

Title: Religion Helps Protect Against 'Deaths of Despair': Study
Category: Health News
Created: 5/6/2020 12:00:00 AM
Last Editorial Review: 5/7/2020 12:00:00 AM

Title: Blood Pressure Dips Upon Standing Might Not Be as Dangerous as Thought
Category: Health News
Created: 1/28/2020 12:00:00 AM
Last Editorial Review: 1/29/2020 12:00:00 AM

Title: AHA News: These Stroke Survivors May Not Be Prescribed Enough Blood Pressure Meds
Category: Health News
Created: 2/21/2020 12:00:00 AM
Last Editorial Review: 2/24/2020 12:00:00 AM

Title: Low Blood Pressure (Hypotension)
Category: Diseases and Conditions
Created: 12/31/1997 12:00:00 AM
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Title: 'Couch Potato' Lifestyle Poses Danger to Women's Hearts
Category: Health News
Created: 2/18/2020 12:00:00 AM
Last Editorial Review: 2/19/2020 12:00:00 AM

Title: Birth Control Pill vs. Shot (Depo-Provera): Similarities and Differences
Category: Diseases and Conditions
Created: 6/15/2017 12:00:00 AM
Last Editorial Review: 4/13/2020 12:00:00 AM

Title: AHA News: Make Mother's Day Last All Year With Wellness and Appreciation
Category: Health News
Created: 4/29/2020 12:00:00 AM
Last Editorial Review: 4/30/2020 12:00:00 AM

Title: One Dose of HPV Vaccine May Protect Against Cervical Cancer
Category: Health News
Created: 2/10/2020 12:00:00 AM
Last Editorial Review: 2/10/2020 12:00:00 AM

Title: More Hot Flashes Could Mean Higher Odds for Heart Trouble
Category: Health News
Created: 9/24/2019 12:00:00 AM
Last Editorial Review: 9/24/2019 12:00:00 AM

Title: Health Tip: Relieving Hot Flashes
Category: Health News
Created: 10/4/2019 12:00:00 AM
Last Editorial Review: 10/4/2019 12:00:00 AM

Title: Pot Use Among U.S. Seniors Nearly Doubled in 3 Years
Category: Health News
Created: 2/24/2020 12:00:00 AM
Last Editorial Review: 2/25/2020 12:00:00 AM

Title: Welcome to the 'Smart Toilet' That Can Spot Disease
Category: Health News
Created: 4/17/2020 12:00:00 AM
Last Editorial Review: 4/17/2020 12:00:00 AM

Title: Got Flu? Deal Quickly With Complications
Category: Health News
Created: 2/2/2020 12:00:00 AM
Last Editorial Review: 2/3/2020 12:00:00 AM

Title: azithromycin (Zithromax): Potential COVID-19 Combo Drug
Category: Medications
Created: 12/31/1997 12:00:00 AM
Last Editorial Review: 3/30/2020 12:00:00 AM

Title: Dangerously Hot Days for U.S. Farm Workers Could Double by 2050
Category: Health News
Created: 5/5/2020 12:00:00 AM
Last Editorial Review: 5/6/2020 12:00:00 AM

Title: Fewer Kids in Cancer Trials, Which Might Not Be a Bad Thing
Category: Health News
Created: 5/5/2020 12:00:00 AM
Last Editorial Review: 5/6/2020 12:00:00 AM

Title: Traveling Abroad? Make Sure Your Measles Shot Is Up to Date
Category: Health News
Created: 7/24/2019 12:00:00 AM
Last Editorial Review: 7/24/2019 12:00:00 AM

Title: Using Pot to Help You Sleep? It Could Backfire
Category: Health News
Created: 4/14/2020 12:00:00 AM
Last Editorial Review: 4/15/2020 12:00:00 AM

This first-in-human phase I study evaluated the pharmacokinetics, safety, and preliminary efficacy of telisotuzumab, formerly called ABT-700, an antagonistic antibody directed against c-Met. For dose escalation (3+3 design), 3 to 6 patients with advanced solid tumors were enrolled into four dose cohorts (5–25 mg/kg). In the dose-expansion phase, a subset of patients was prospectively selected for MET amplification (FISH screening). Patients received telisotuzumab intravenously on day 1 every 21 days. For dose expansion, 15 mg/kg was chosen as the dose on the basis of safety, pharmacokinetics, and other data from the escalation cohorts. Forty-five patients were enrolled and received at least one dose of telisotuzumab (dose escalation, n = 15; dose expansion, n = 30). Telisotuzumab showed a linear pharmacokinetics profile; peak plasma concentration was proportional to dose level. There were no acute infusion reactions and no dose-limiting toxicities were observed. The most common treatment-related adverse events included hypoalbuminemia (n = 9, 20.0%) and fatigue (n = 5, 11.1%). By Response Evaluation Criteria In Solid Tumors (RECIST), 4 of 10 (40.0%) patients with MET-amplified tumors had confirmed partial response in target lesions (one ovarian, two gastric, and one esophageal), two (20.0%) had stable disease, three (30.0%) had progressive disease; one patient was unable to be evaluated. Among patients with nonamplified tumors (n = 35), no objective responses were observed; however, 11 patients had stable disease per RECIST criteria. In conclusion, telisotuzumab has an acceptable safety profile with clinical activity observed in patients with MET-amplified advanced solid tumors.

Overexpression of transcription factor 3 in alveolar soft part sarcoma(ASPS) results in upregulation of cell proliferation pathways. No standard treatment algorithm exists for ASPS; multikinase inhibitors[tyrosine kinase inhibitor (TKI)] and immune checkpoint inhibitors (ICI) have shown clinical benefit. To date, no studies have reported on management strategies or sequencing of therapy. We evaluated ASPS treatment patterns and responses in an experimental therapeutics clinic. Genomic and morphoproteomic analysis was performed to further elucidate novel targets. We retrospectively reviewed patients with ASPS treated on clinical trials. Demographic and clinical next-generation sequencing (NGS) profiles were collected. AACR GENIE database was queried to further evaluate aberrations in ASPS. Morphoproteomic analysis was carried out to better define the biology of ASPS with integration of genomic and proteomic findings. Eleven patients with ASPS were identified; 7 received NGS testing and mutations in CDKN2A (n = 1) and hepatocyte growth factor (n = 1) were present. Ten patients were treated with TKIs with stable disease as best response and 4 patients with ICI (three partial responses). Within GENIE, 20 patients were identified harboring 3 called pathogenic mutations. Tumor mutation burden was low in all samples. Morphoproteomic analysis confirmed the expression of phosphorylated c-Met. In addition, fatty acid synthase and phosphorylated-STAT3 were detected in tumor cell cytoplasm and nuclei. Patients with ASPS have a quiescent genome and derive clinical benefit from VEGF-targeting TKIs. Morphoproteomic analysis has provided both additional correlative pathways and angiogenic mechanisms that are targetable for patients with ASPS. Our study suggests that sequential therapy with TKIs and immune checkpoint inhibitors is a reasonable management strategy.

Expression of the DNA/RNA helicase schlafen family member 11 (SLFN11) has been identified as a sensitizer of tumor cells to DNA-damaging agents including platinum chemotherapy. We assessed the impact of SLFN11 expression on response to platinum chemotherapy and outcomes in patients with metastatic castration-resistant prostate cancer (CRPC). Tumor expression of SLFN11 was assessed in 41 patients with CRPC treated with platinum chemotherapy by RNA sequencing (RNA-seq) of metastatic biopsy tissue (n = 27) and/or immunofluorescence in circulating tumor cells (CTC; n = 20). Cox regression and Kaplan–Meier methods were used to evaluate the association of SLFN11 expression with radiographic progression-free survival (rPFS) and overall survival (OS). Multivariate analysis included tumor histology (i.e., adenocarcinoma or neuroendocrine) and the presence or absence of DNA repair aberrations. Patient-derived organoids with SLFN11 expression and after knockout by CRISPR-Cas9 were treated with platinum and assessed for changes in dose response. Patients were treated with platinum combination (N = 38) or platinum monotherapy (N = 3). Median lines of prior therapy for CRPC was two. Median OS was 8.7 months. Overexpression of SLFN11 in metastatic tumors by RNA-seq was associated with longer rPFS compared with those without overexpression (6.9 vs. 2.8 months, HR = 3.72; 95% confidence interval (CI), 1.56–8.87; P < 0.001); similar results were observed for patients with SLFN11-positive versus SLFN11-negative CTCs (rPFS 6.0 vs. 2.2 months, HR = 4.02; 95% CI, 0.77–20.86; P = 0.002). A prostate-specific antigen (PSA) decline of ≥50% was observed in all patients with SLFN11 overexpression. No association was observed between SLFN11 expression and OS. On multivariable analysis, SLFN11 was an independent factor associated with rPFS on platinum therapy. Platinum response of organoids expressing SLFN11 was reduced after SLFN11 knockout. Our data suggest that SLFN11 expression might identify patients with CRPC with a better response to platinum chemotherapy independent of histology or other genomic alterations. Additional studies, also in the context of PARP inhibitors, are warranted.

Dysregulation of DNA methylation is an established feature of breast cancers. DNA demethylating therapies like decitabine are proposed for the treatment of triple-negative breast cancers (TNBC) and indicators of response need to be identified. For this purpose, we characterized the effects of decitabine in a panel of 10 breast cancer cell lines and observed a range of sensitivity to decitabine that was not subtype specific. Knockdown of potential key effectors demonstrated the requirement of deoxycytidine kinase (DCK) for decitabine response in breast cancer cells. In treatment-naïve breast tumors, DCK was higher in TNBCs, and DCK levels were sustained or increased post chemotherapy treatment. This suggests that limited DCK levels will not be a barrier to response in patients with TNBC treated with decitabine as a second-line treatment or in a clinical trial. Methylome analysis revealed that genome-wide, region-specific, tumor suppressor gene–specific methylation, and decitabine-induced demethylation did not predict response to decitabine. Gene set enrichment analysis of transcriptome data demonstrated that decitabine induced genes within apoptosis, cell cycle, stress, and immune pathways. Induced genes included those characterized by the viral mimicry response; however, knockdown of key effectors of the pathway did not affect decitabine sensitivity suggesting that breast cancer growth suppression by decitabine is independent of viral mimicry. Finally, taxol-resistant breast cancer cells expressing high levels of multidrug resistance transporter ABCB1 remained sensitive to decitabine, suggesting that the drug could be used as second-line treatment for chemoresistant patients.

Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft tumors led to an increased number of persistent DSBs. Oral administration of M3814 to two xenograft models of human cancer, using a clinically established 6-week fractionated radiation schedule, strongly potentiated the antitumor activity of IR and led to complete tumor regression at nontoxic doses. Our results strongly support DNA-PK inhibition as a novel approach for the combination radiotherapy of cancer. M3814 is currently under investigation in combination with radiotherapy in clinical trials.

Purpose: The purpose of this study was to determine strength of muscles involved with instrumentation (scaling) by dental hygienists and the additive effects of cellular (mobile) phone usage, as indicated by measurements of muscular force generation.Methods: A convenience sample of licensed dental hygienists currently in clinical practice (n=16) and an equal number of individuals not currently using devices/tools repetitively for work (n=16), agreed to participate in this pilot study. All participants completed a modified cell phone usage questionnaire to determine their use pattern and frequency. Upon completion of the questionnaire, participants' force production in six muscle groups was measured using a hand-held dynamometer. Descriptive statistics were used to analyze the data.Results: A total of 16 licensed dental hygienists (n=16) and 16 participants with no history of using tools/devices repetitively for work (n=16), comprised the experimental and control groups, repectively. The control group generated greater muscle force than the experimental group for the abductor pollicis longus (p=0.045). Significant differences were identified when comparing the low mobile phone users in the experimental group to the control group for the flexor pollicis brevis (p=0.031), abductor pollicis longus (p=0.031), and flexor digitorum (p=0.006), with the control group demonstrating higher muscle force. Years in clinical practice and mobile phone use was shown to have a significant effect on muscular force generation for the flexor pollicis brevis (F=3.645, df=3, p=0.020) and flexor digitorum (F=3.560, df=3, p=0.022); subjects who practiced dental hygiene the longest produced the least amount of muscle force.Conclusion: Results from this pilot study indicate there are no significant additive effects of cell phone use and dental hygiene practice on finger muscles used for instrumentation. However, results indicate that dental hygiene practice demonstrated significant effects on muscular strength as compared to individuals who do not use tools/devices repetitively for work. The small sample size may have impacted results and the study should be repeated with a larger sample.

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (^–/–) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.­

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R^–/–) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by ²H/¹³C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) establishes lifelong infections in humans, a process that relies on its ability to thwart innate and adaptive immune defenses of the host. Recently, we reported that HIV-1 infection results in a dramatic reduction of the cellular peroxisome pool. Peroxisomes are metabolic organelles that also function as signaling platforms in the innate immune response. Here, we show that the HIV-1 accessory protein Vpu is necessary and sufficient for the depletion of cellular peroxisomes during infection. Vpu induces the expression of four microRNAs that target mRNAs encoding proteins required for peroxisome formation and metabolic function. The ability of Vpu to downregulate peroxisomes was found to be dependent upon the Wnt/β-catenin signaling pathway. Given the importance of peroxisomes in innate immune signaling and central nervous system function, the roles of Vpu in dampening antiviral signaling appear to be more diverse than previously realized. Finally, our findings highlight a potential role for Wnt/β-catenin signaling in peroxisome homeostasis through modulating the production of biogenesis factors.

IMPORTANCE People living with HIV can experience accelerated aging and the development of neurological disorders. Recently, we reported that HIV-1 infection results in a dramatic loss of peroxisomes in macrophages and brain tissue. This is significant because (i) peroxisomes are important for the innate immune response and (ii) loss of peroxisome function is associated with cellular aging and neurodegeneration. Accordingly, understanding how HIV-1 infection causes peroxisome depletion may provide clues regarding how the virus establishes persistent infections and, potentially, the development of neurological disorders. Here, we show that the accessory protein Vpu is necessary and sufficient for the induction of microRNAs that target peroxisome biogenesis factors. The ability of Vpu to downregulate peroxisome formation depends on the Wnt/β-catenin pathway. Thus, in addition to revealing a novel mechanism by which HIV-1 uses intracellular signaling pathways to target antiviral signaling platforms (peroxisomes), we have uncovered a previously unknown link between the Wnt/β-catenin pathway and peroxisome homeostasis.

ABSTRACT

Obesity is associated with increased disease severity, elevated viral titers in exhaled breath, and significantly prolonged viral shed during influenza A virus infection. Due to the mutable nature of RNA viruses, we questioned whether obesity could also influence influenza virus population diversity. Here, we show that minor variants rapidly emerge in obese mice. The variants exhibit increased viral replication, resulting in enhanced virulence in wild-type mice. The increased diversity of the viral population correlated with decreased type I interferon responses, and treatment of obese mice with recombinant interferon reduced viral diversity, suggesting that the delayed antiviral response exhibited in obesity permits the emergence of a more virulent influenza virus population. This is not unique to obese mice. Obesity-derived normal human bronchial epithelial (NHBE) cells also showed decreased interferon responses and increased viral replication, suggesting that viral diversity also was impacted in this increasing population.

IMPORTANCE Currently, 50% of the adult population worldwide is overweight or obese. In these studies, we demonstrate that obesity not only enhances the severity of influenza infection but also impacts viral diversity. The altered microenvironment associated with obesity supports a more diverse viral quasispecies and affords the emergence of potentially pathogenic variants capable of inducing greater disease severity in lean hosts. This is likely due to the impaired interferon response, which is seen in both obese mice and obesity-derived human bronchial epithelial cells, suggesting that obesity, aside from its impact on influenza virus pathogenesis, permits the stochastic accumulation of potentially pathogenic viral variants, raising concerns about its public health impact as the prevalence of obesity continues to rise.

ABSTRACT

The synergy between Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) interferes with therapy and facilitates the pathogenesis of both human pathogens. Fundamental mechanisms by which M. tuberculosis exacerbates HIV-1 infection are not clear. Here, we show that exosomes secreted by macrophages infected with M. tuberculosis, including drug-resistant clinical strains, reactivated HIV-1 by inducing oxidative stress. Mechanistically, M. tuberculosis-specific exosomes realigned mitochondrial and nonmitochondrial oxygen consumption rates (OCR) and modulated the expression of host genes mediating oxidative stress response, inflammation, and HIV-1 transactivation. Proteomics analyses revealed the enrichment of several host factors (e.g., HIF-1α, galectins, and Hsp90) known to promote HIV-1 reactivation in M. tuberculosis-specific exosomes. Treatment with a known antioxidant—N-acetyl cysteine (NAC)—or with inhibitors of host factors—galectins and Hsp90—attenuated HIV-1 reactivation by M. tuberculosis-specific exosomes. Our findings uncover new paradigms for understanding the redox and bioenergetics bases of HIV-M. tuberculosis coinfection, which will enable the design of effective therapeutic strategies.

IMPORTANCE Globally, individuals coinfected with the AIDS virus (HIV-1) and with M. tuberculosis (causative agent of tuberculosis [TB]) pose major obstacles in the clinical management of both diseases. At the heart of this issue is the apparent synergy between the two human pathogens. On the one hand, mechanisms induced by HIV-1 for reactivation of TB in AIDS patients are well characterized. On the other hand, while clinical findings clearly identified TB as a risk factor for HIV-1 reactivation and associated mortality, basic mechanisms by which M. tuberculosis exacerbates HIV-1 replication and infection remain poorly characterized. The significance of our research is in identifying the role of fundamental mechanisms such as redox and energy metabolism in catalyzing HIV-M. tuberculosis synergy. The quantification of redox and respiratory parameters affected by M. tuberculosis in stimulating HIV-1 will greatly enhance our understanding of HIV-M. tuberculosis coinfection, leading to a wider impact on the biomedical research community and creating new translational opportunities.

ABSTRACT

Bacterial flagella are rotating nanomachines required for motility. Flagellar gene expression and protein secretion are coordinated for efficient flagellar biogenesis. Polar flagellates, unlike peritrichous bacteria, commonly order flagellar rod and hook gene transcription as a separate step after production of the MS ring, C ring, and flagellar type III secretion system (fT3SS) core proteins that form a competent fT3SS. Conserved regulatory mechanisms in diverse polar flagellates to create this polar flagellar transcriptional program have not been thoroughly assimilated. Using in silico and genetic analyses and our previous findings in Campylobacter jejuni as a foundation, we observed a large subset of Gram-negative bacteria with the FlhF/FlhG regulatory system for polar flagellation to possess flagellum-associated two-component signal transduction systems (TCSs). We present data supporting a general theme in polar flagellates whereby MS ring, rotor, and fT3SS proteins contribute to a regulatory checkpoint during polar flagellar biogenesis. We demonstrate that Vibrio cholerae and Pseudomonas aeruginosa require the formation of this regulatory checkpoint for the TCSs to directly activate subsequent rod and hook gene transcription, which are hallmarks of the polar flagellar transcriptional program. By reprogramming transcription in V. cholerae to more closely follow the peritrichous flagellar transcriptional program, we discovered a link between the polar flagellar transcription program and the activity of FlhF/FlhG flagellar biogenesis regulators in which the transcriptional program allows polar flagellates to continue to produce flagella for motility when FlhF or FlhG activity may be altered. Our findings integrate flagellar transcriptional and biogenesis regulatory processes involved in polar flagellation in many species.

IMPORTANCE Relative to peritrichous bacteria, polar flagellates possess regulatory systems that order flagellar gene transcription differently and produce flagella in specific numbers only at poles. How transcriptional and flagellar biogenesis regulatory systems are interlinked to promote the correct synthesis of polar flagella in diverse species has largely been unexplored. We found evidence for many Gram-negative polar flagellates encoding two-component signal transduction systems with activity linked to the formation of flagellar type III secretion systems to enable production of flagellar rod and hook proteins at a discrete, subsequent stage during flagellar assembly. This polar flagellar transcriptional program assists, in some manner, the FlhF/FlhG flagellar biogenesis regulatory system, which forms specific flagellation patterns in polar flagellates in maintaining flagellation and motility when activity of FlhF or FlhG might be altered. Our work provides insight into the multiple regulatory processes required for polar flagellation.

ABSTRACT

In Escherichia coli, the chemotaxis response regulator CheY-P binds to FliM, a component of the switch complex at the base of the bacterial flagellar motor, to modulate the direction of motor rotation. The bacterial flagellar motor is ultrasensitive to the concentration of unbound CheY-P in the cytoplasm. CheY-P binds to FliM molecules both in the cytoplasm and on the motor. As the concentration of FliM unavoidably varies from cell to cell, leading to a variation of unbound CheY-P concentration in the cytoplasm, this raises the question whether the flagellar motor is robust against this variation, that is, whether the rotational bias of the motor is more or less constant as the concentration of FliM varies. Here, we showed that the motor is robust against variations of the concentration of FliM. We identified adaptive remodeling of the motor as the mechanism for this robustness. As the level of FliM molecules changes, resulting in different amounts of the unbound CheY-P molecules, the motor adaptively changes the composition of its switch complex to compensate for this effect.

IMPORTANCE The bacterial flagellar motor is an ultrasensitive motor. Its output, the probability of the motor turning clockwise, depends sensitively on the occupancy of the protein FliM (a component on the switch complex of the motor) by the input CheY-P molecules. With a limited cellular pool of CheY-P molecules, cell-to-cell variation of the FliM level would lead to large unwanted variation of the motor output if not compensated. Here, we showed that the motor output is robust against the variation of FliM level and identified the adaptive remodeling of the motor switch complex as the mechanism for this robustness.

ABSTRACT

Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.

IMPORTANCE Pneumocystis continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunodepleting monoclonal antibodies. Annual health care associated with Pneumocystis pneumonia costs ~$475 million dollars in the United States alone. In addition to causing overt disease in immunodeficient individuals, Pneumocystis can cause subclinical infection or colonization in healthy individuals, which may play an important role in species preservation and disease transmission. Our work sheds new light on the diversity and complexity of the msg superfamily and strongly suggests that the versatility of this superfamily reflects multiple functions, including antigenic variation to allow immune evasion and optimal adaptation to host environmental conditions to promote efficient infection and transmission. These findings are essential to consider in developing new diagnostic and therapeutic strategies.

ABSTRACT

Cryptosporidium parvum and Cryptosporidium hominis have emerged as major enteric pathogens of infants in the developing world, in addition to their known importance in immunocompromised adults. Although there has been recent progress in identifying new small molecules that inhibit Cryptosporidium sp. growth in vitro or in animal models, we lack information about their mechanism of action, potency across the life cycle, and cidal versus static activities. Here, we explored four potent classes of compounds that include inhibitors that likely target phosphatidylinositol 4 kinase (PI4K), phenylalanine-tRNA synthetase (PheRS), and several potent inhibitors with unknown mechanisms of action. We utilized monoclonal antibodies and gene expression probes for staging life cycle development to define the timing of when inhibitors were active during the life cycle of Cryptosporidium parvum grown in vitro. These different classes of inhibitors targeted different stages of the life cycle, including compounds that blocked replication (PheRS inhibitors), prevented the segmentation of daughter cells and thus blocked egress (PI4K inhibitors), or affected sexual-stage development (a piperazine compound of unknown mechanism). Long-term cultivation of C. parvum in epithelial cell monolayers derived from intestinal stem cells was used to distinguish between cidal and static activities based on the ability of parasites to recover from treatment. Collectively, these approaches should aid in identifying mechanisms of action and for designing in vivo efficacy studies based on time-dependent concentrations needed to achieve cidal activity.

IMPORTANCE Currently, nitazoxanide is the only FDA-approved treatment for cryptosporidiosis; unfortunately, it is ineffective in immunocompromised patients, has varied efficacy in immunocompetent individuals, and is not approved in infants under 1 year of age. Identifying new inhibitors for the treatment of cryptosporidiosis requires standardized and quantifiable in vitro assays for assessing potency, selectivity, timing of activity, and reversibility. Here, we provide new protocols for defining which stages of the life cycle are susceptible to four highly active compound classes that likely inhibit different targets in the parasite. We also utilize a newly developed long-term culture system to define assays for monitoring reversibility as a means of defining cidal activity as a function of concentration and time of treatment. These assays should provide valuable in vitro parameters to establish conditions for efficacious in vivo treatment.

ABSTRACT

Anti-galactose-α-1,3-galactose (anti-α-Gal) antibody is naturally expressed at a high level in humans. It constitutes about 1% of immunoglobulins found in human blood. Here, we designed a live attenuated influenza virus vaccine that can generate α-Gal epitopes in infected cells in order to facilitate opsonization of infected cells, thereby enhancing vaccine-induced immune responses. In the presence of normal human sera, cells infected with this mutant can enhance phagocytosis of human macrophages and cytotoxicity of NK cells in vitro. Using a knockout mouse strain that allows expression of anti-α-Gal antibody in vivo, we showed that this strategy can increase vaccine immunogenicity and the breadth of protection. This vaccine can induce 100% protection against a lethal heterosubtypic group 1 (H5) or group 2 (mouse-adapted H3) influenza virus challenge in the mouse model. In contrast, its heterosubtypic protective effect in wild-type or knockout mice that do not have anti-α-Gal antibody expression is only partial, demonstrating that the enhanced vaccine-induced protection requires anti-α-Gal antibody upon vaccination. Anti-α-Gal-expressing knockout mice immunized with this vaccine produce robust humoral and cell-mediated responses upon a lethal virus challenge. This vaccine can stimulate CD11b^lo/– pulmonary dendritic cells, which are known to be crucial for clearance of influenza virus. Our approach provides a novel strategy for developing next-generation influenza virus vaccines.

IMPORTANCE Influenza A viruses have multiple HA subtypes that are antigenically diverse. Classical influenza virus vaccines are subtype specific, and they cannot induce satisfactory heterosubtypic immunity against multiple influenza virus subtypes. Here, we developed a live attenuated H1N1 influenza virus vaccine that allows the expression of α-Gal epitopes by infected cells. Anti-α-Gal antibody is naturally produced by humans. In the presence of this antibody, human cells infected with this experimental vaccine virus can enhance several antibody-mediated immune responses in vitro. Importantly, mice expressing anti-α-Gal antibody in vivo can be fully protected by this H1N1 vaccine against a lethal H5 or H3 virus challenge. Our work demonstrates a new strategy for using a single influenza virus strain to induce broadly cross-reactive immune responses against different influenza virus subtypes.

ABSTRACT

The cell cycle is a critical component of cellular proliferation, differentiation, and response to stress, yet its role in the regulation of intracellular symbioses is not well understood. To explore host-symbiont cell cycle coordination in a marine symbiosis, we employed a model for coral-dinoflagellate associations: the tropical sea anemone Aiptasia (Exaiptasia pallida) and its native microalgal photosymbionts (Breviolum minutum and Breviolum psygmophilum). Using fluorescent labeling and spatial point-pattern image analyses to characterize cell population distributions in both partners, we developed protocols that are tailored to the three-dimensional cellular landscape of a symbiotic sea anemone tentacle. Introducing cultured symbiont cells to symbiont-free adult hosts increased overall host cell proliferation rates. The acceleration occurred predominantly in the symbiont-containing gastrodermis near clusters of symbionts but was also observed in symbiont-free epidermal tissue layers, indicating that the presence of symbionts contributes to elevated proliferation rates in the entire host during colonization. Symbiont cell cycle progression differed between cultured algae and those residing within hosts; the endosymbiotic state resulted in increased S-phase but decreased G₂/M-phase symbiont populations. These phenotypes and the deceleration of cell cycle progression varied with symbiont identity and host nutritional status. These results demonstrate that host and symbiont cells have substantial and species-specific effects on the proliferation rates of their mutualistic partners. This is the first empirical evidence to support species-specific regulation of the symbiont cell cycle within a single cnidarian-dinoflagellate association; similar regulatory mechanisms likely govern interpartner coordination in other coral-algal symbioses and shape their ecophysiological responses to a changing climate.

IMPORTANCE Biomass regulation is critical to the overall health of cnidarian-dinoflagellate symbioses. Despite the central role of the cell cycle in the growth and proliferation of cnidarian host cells and dinoflagellate symbionts, there are few studies that have examined the potential for host-symbiont coregulation. This study provides evidence for the acceleration of host cell proliferation when in local proximity to clusters of symbionts within cnidarian tentacles. The findings suggest that symbionts augment the cell cycle of not only their enveloping host cells but also neighboring cells in the epidermis and gastrodermis. This provides a possible mechanism for rapid colonization of cnidarian tissues. In addition, the cell cycles of symbionts differed depending on nutritional regime, symbiotic state, and species identity. The responses of cell cycle profiles to these different factors implicate a role for species-specific regulation of symbiont cell cycles within host cnidarian tissues.

ABSTRACT

Over the past decade, Candida auris has emerged as an urgent threat to public health. Initially reported from cases of ear infections in Japan and Korea, C. auris has since been detected around the world. While whole-genome sequencing has been extensively used to trace the genetic relationships of the global emergence and local outbreaks, a recent report in mBio describes a targeted genotyping method as a rapid and inexpensive method for classifying C. auris isolates (T. de Groot, Y. Puts, I. Berrio, A. Chowdhary, and J. F. Meis, mBio 11:e02971-19, https://doi.org/10.1128/mBio.02971-19, 2020).

ABSTRACT

The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3' untranslated regions (UTRs). The umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3' UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant’s vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana, p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3' UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor.

IMPORTANCE Nonsense-mediated decay (NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3' untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.

ABSTRACT

During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. Here, we identified a novel cis-acting sequence (5'-GUA AAG CUA CGG GAC GGU-3'), intronic splicing enhancer 3 (ISE3), which lies 72 nucleotides upstream of the second splice acceptor (A2-2) site of the second intron that defines the exon of the mRNA encoding the 11-kDa viral nonstructural protein. RNA binding motif protein 45 (RBM45) specifically binds to ISE3 with high affinity (equilibrium dissociation constant [K_D] = 33 nM) mediated by its RNA recognition domain and 2-homo-oligomer assembly domain (RRM2-HOA). Knockdown of RBM45 expression or ectopic overexpression of RRM2-HOA in human erythroid progenitor cells (EPCs) expanded ex vivo significantly decreased the level of viral mRNA spliced at the A2-2 acceptor but not that of the mRNA spliced at A2-1 that encodes VP2. Moreover, silent mutations of ISE3 in an infectious DNA of B19V significantly reduced 11-kDa expression. Notably, RBM45 also specifically interacts in vitro with ISE2, which shares the octanucleotide (GGGACGGU) with ISE3. Taken together, our results suggest that RBM45, through binding to both ISE2 and ISE3, is an essential host factor for maturation of 11-kDa-encoding mRNA.

IMPORTANCE Human parvovirus B19 (B19V) is a human pathogen that causes severe hematological disorders in immunocompromised individuals. B19V infection has a remarkable tropism with respect to human erythroid progenitor cells (EPCs) in human bone marrow and fetal liver. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. Our studies revealed that a cellular RNA binding protein, RBM45, binds to two intron splicing enhancers and is essential for the maturation of the small nonstructural protein 11-kDa-encoding mRNA. The 11-kDa protein plays an important role not only in B19V infection-induced apoptosis but also in viral DNA replication. Thus, the identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders.

ABSTRACT

Although the pathogen recognition receptor pathways that activate cell-intrinsic antiviral responses are well delineated, less is known about how the host regulates this response to prevent sustained signaling and possible immune-mediated damage. Using a genome-wide CRISPR-Cas9 screening approach to identify host factors that modulate interferon-stimulated gene (ISG) expression, we identified the DNA binding protein Barrier-to-autointegration factor 1 (Banf1), a previously described inhibitor of retrovirus integration, as a modulator of basal cell-intrinsic immunity. Ablation of Banf1 by gene editing resulted in chromatin activation near host defense genes with associated increased expression of ISGs, including Oas2, Rsad2 (viperin), Ifit1, and ISG15. The phenotype in Banf1-deficient cells occurred through a cGAS-, STING-, and IRF3-dependent signaling axis, was associated with reduced infection of RNA and DNA viruses, and was reversed in Banf1 complemented cells. Confocal microscopy and biochemical studies revealed that a loss of Banf1 expression resulted in higher level of cytosolic double-stranded DNA at baseline. Our study identifies an undescribed role for Banf1 in regulating the levels of cytoplasmic DNA and cGAS-dependent ISG homeostasis and suggests possible therapeutic directions for promoting or inhibiting cell-intrinsic innate immune responses.

IMPORTANCE Although the interferon (IFN) signaling pathway is a key host mechanism to restrict infection of a diverse range of viral pathogens, its unrestrained activity either at baseline or in the context of an immune response can result in host cell damage and injury. Here, we used a genome-wide CRISPR-Cas9 screen and identified the DNA binding protein Barrier-to-autointegration factor 1 (Banf1) as a modulator of basal cell-intrinsic immunity. A loss of Banf1 expression resulted in higher level of cytosolic double-stranded DNA at baseline, which triggered IFN-stimulated gene expression via a cGAS-STING-IRF3 axis that did not require type I IFN or STAT1 signaling. Our experiments define a regulatory network in which Banf1 limits basal inflammation by preventing self DNA accumulation in the cytosol.