Kaposi sarcoma-associated herpesvirus (KSHV) is necessary but not sufficient for primary effusion lymphoma (PEL) development. Alterations in cellular signaling pathways are also a characteristic of PEL. Other B cell lymphomas have acquired an oncogenic mutation in the myeloid differentiation primary response 88 (MYD88) gene. The MYD88 L265P mutant results in the activation of interleukin-1 receptor associated kinase (IRAK). To probe IRAK/MYD88 signaling in PEL, we employed CRISPR/Cas9 technology to generate stable deletion clones in BCBL-1Cas9 and BC-1Cas9 cells. To look for off-target effects, we determined the complete exome of the BCBL-1Cas9 and BC-1Cas9 cells. Deletion of either MYD88, IRAK4, or IRAK1 abolished interleukin-1 beta (IL-1β) signaling; however, we were able to grow stable subclones from each population. Transcriptome sequencing (RNA-seq) analysis of IRAK4 knockout cell lines (IRAK4 KOs) showed that the IRAK pathway induced cellular signals constitutively, independent of IL-1β stimulation, which was abrogated by deletion of IRAK4. Transient complementation with IRAK1 increased NF-B activity in MYD88 KO, IRAK1 KO, and IRAK4 KO cells even in the absence of IL-1β. IL-10, a hallmark of PEL, was dependent on the IRAK pathway, as IRAK4 KOs showed reduced IL-10 levels. We surmise that, unlike B cell receptor (BCR) signaling, MYD88/IRAK signaling is constitutively active in PEL, but that under cell culture conditions, PEL rapidly became independent of this pathway.

IMPORTANCE One hundred percent of primary effusion lymphoma (PEL) cases are associated with Kaposi sarcoma-associated herpesvirus (KSHV). PEL cell lines, such as BCBL-1, are the workhorse for understanding this human oncovirus and the host pathways that KSHV dysregulates. Understanding their function is important for developing new therapies as well as identifying high-risk patient groups. The myeloid differentiation primary response 88 (MYD88)/interleukin-1 receptor associated kinase (IRAK) pathway, which has progrowth functions in other B cell lymphomas, has not been fully explored in PEL. By performing CRISPR/Cas9 knockout (KO) studies targeting the IRAK pathway in PEL, we were able to determine that established PEL cell lines can circumvent the loss of IRAK1, IRAK4, and MYD88; however, the deletion clones are deficient in interleukin-10 (IL-10) production. Since IL-10 suppresses T cell function, this suggests that the IRAK pathway may serve a function in vivo and during early-stage development of PEL.

The TEAD family of transcription factors requires associating cofactors to induce gene expression. TEAD1 is known to activate the early promoter of human papillomavirus (HPV), but the precise mechanisms of TEAD1-mediated transactivation of the HPV promoter, including its relevant cofactors, remain unexplored. Here, we reveal that VGLL1, a TEAD-interacting cofactor, contributes to HPV early gene expression. Knockdown of VGLL1 and/or TEAD1 led to a decrease in viral early gene expression in human cervical keratinocytes and cervical cancer cell lines. We identified 11 TEAD1 target sites in the HPV16 long control region (LCR) by in vitro DNA pulldown assays; 8 of these sites contributed to the transcriptional activation of the early promoter in luciferase reporter assays. VGLL1 bound to the HPV16 LCR via its interaction with TEAD1 both in vitro and in vivo. Furthermore, introducing HPV16 and HPV18 whole genomes into primary human keratinocytes led to increased levels of VGLL1, due in part to the upregulation of TEADs. These results suggest that multiple VGLL1/TEAD1 complexes are recruited to the LCR to support the efficient transcription of HPV early genes.

IMPORTANCE Although a number of transcription factors have been reported to be involved in HPV gene expression, little is known about the cofactors that support HPV transcription. In this study, we demonstrate that the transcriptional cofactor VGLL1 plays a prominent role in HPV early gene expression, dependent on its association with the transcription factor TEAD1. Whereas TEAD1 is ubiquitously expressed in a variety of tissues, VGLL1 displays tissue-specific expression and is implicated in the development and differentiation of epithelial lineage tissues, where HPV gene expression occurs. Our results suggest that VGLL1 may contribute to the epithelial specificity of HPV gene expression, providing new insights into the mechanisms that regulate HPV infection. Further, VGLL1 is also critical for the growth of cervical cancer cells and may represent a novel therapeutic target for HPV-associated cancers.

Although substantial progress has been made in depicting the molecular pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection, the comprehensive mechanism of HIV-1 latency and the most promising therapeutic strategies to effectively reactivate the HIV-1 latent reservoir to achieve a functional cure for AIDS remain to be systematically illuminated. Here, we demonstrated that piwi (P element-induced Wimpy)-like RNA-mediated gene silencing 4 (PIWIL4) played an important role in suppressing HIV-1 transcription and contributed to the latency state in HIV-1-infected cells through its recruitment of various suppressive factors, including heterochromatin protein 1α/β/, SETDB1, and HDAC4. The knockdown of PIWIL4 enhanced HIV-1 transcription and reversed HIV-1 latency in both HIV-1 latently infected Jurkat T cells and primary CD4⁺ T lymphocytes and resting CD4⁺ T lymphocytes from HIV-1-infected individuals on suppressive combined antiretroviral therapy (cART). Furthermore, in the absence of PIWIL4, HIV-1 latently infected Jurkat T cells were more sensitive to reactivation with vorinostat (suberoylanilide hydroxamic acid, or SAHA), JQ1, or prostratin. These findings indicated that PIWIL4 promotes HIV-1 latency by imposing repressive marks at the HIV-1 5' long terminal repeat. Thus, the manipulation of PIWIL4 could be a novel strategy for developing promising latency-reversing agents (LRAs).

IMPORTANCE HIV-1 latency is systematically modulated by host factors and viral proteins. During this process, the suppression of HIV-1 transcription plays an essential role in promoting HIV-1 latency. In this study, we found that PIWIL4 repressed HIV-1 promoter activity and maintained HIV-1 latency. In particular, we report that PIWIL4 can regulate gene expression through its association with the suppressive activity of HDAC4. Therefore, we have identified a new function for PIWIL4: it is not only a suppressor of endogenous retrotransposons but also plays an important role in inhibiting transcription and leading to latent infection of HIV-1, a well-known exogenous retrovirus. Our results also indicate a novel therapeutic target to reactivate the HIV-1 latent reservoir.

The Epstein-Barr virus (EBV) causes human cancers, and epidemiological studies have shown that lytic replication is a risk factor for some of these tumors. This fits with the observation that EBV M81, which was isolated from a Chinese patient with nasopharyngeal carcinoma, induces potent virus production and increases the risk of genetic instability in infected B cells. To find out whether this property extends to viruses found in other parts of the world, we investigated 22 viruses isolated from Western patients. While one-third of the viruses hardly replicated, the remaining viruses showed variable levels of replication, with three isolates replicating at levels close to that of M81 in B cells. We cloned one strongly replicating virus into a bacterial artificial chromosome (BAC); the resulting recombinant virus (MSHJ) retained the properties of its nonrecombinant counterpart and showed similarities to M81, undergoing lytic replication in vitro and in vivo after 3 weeks of latency. In contrast, B cells infected with the nonreplicating Western B95-8 virus showed early but abortive replication accompanied by cytoplasmic BZLF1 expression. Sequencing confirmed that rMSHJ is a Western virus, being genetically much closer to B95-8 than to M81. Spontaneous replication in rM81- and rMSHJ-infected B cells was dependent on phosphorylated Btk and was inhibited by exposure to ibrutinib, opening the way to clinical intervention in patients with abnormal EBV replication. As rMSHJ contains the complete EBV genome and induces lytic replication in infected B cells, it is ideal to perform genetic analyses of all viral functions in Western strains and their associated diseases.

IMPORTANCE The Epstein-Barr virus (EBV) infects the majority of the world population but causes different diseases in different countries. Evidence that lytic replication, the process that leads to new virus progeny, is linked to cancer development is accumulating. Indeed, viruses such as M81 that were isolated from Far Eastern nasopharyngeal carcinomas replicate strongly in B cells. We show here that some viruses isolated from Western patients, including the MSHJ strain, share this property. Moreover, replication of both M81 and of MSHJ was sensitive to ibrutinib, a commonly used drug, thereby opening an opportunity for therapeutic intervention. Sequencing of MSHJ showed that this virus is quite distant from M81 and is much closer to nonreplicating Western viruses. We conclude that Western EBV strains are heterogeneous, with some viruses being able to replicate more strongly and therefore being potentially more pathogenic than others, and that the virus sequence information alone cannot predict this property.

Simian-human immunodeficiency virus (SHIV) infection of rhesus monkeys is an important preclinical model for human immunodeficiency virus type 1 (HIV-1) vaccines, therapeutics, and cure strategies. SHIVs have been optimized by incorporating HIV-1 Env residue 375 mutations that mimic the bulky or hydrophobic residues typically found in simian immunodeficiency virus (SIV) Env to improve rhesus CD4 binding. We applied this strategy to three SHIV challenge stocks (SHIV-SF162p3, SHIV-AE16, and SHIV-325c) and observed three distinct outcomes. We constructed six Env375 variants (M, H, W, Y, F, and S) for each SHIV, and we performed a pool competition study in rhesus monkeys to define the optimal variant for each SHIV prior to generating large-scale challenge stocks. We identified SHIV-SF162p3S/wild type, SHIV-AE16W, and SHIV-325cH as the optimal variants. SHIV-SF162p3S could not be improved, as it already contained the optimal Env375 residue. SHIV-AE16W exhibited a similar replicative capacity to the parental SHIV-AE16 stock. In contrast, SHIV-325cH demonstrated a 2.6-log higher peak and 1.6-log higher setpoint viral loads than the parental SHIV-325c stock. These data demonstrate the diversity of potential outcomes following Env375 modification in SHIVs. Moreover, the clade C SHIV-325cH challenge stock may prove useful for evaluating prophylactic or therapeutic interventions against clade C HIV-1.

IMPORTANCE We sought to enhance the infectivity of three SHIV stocks by optimization of a key residue in human immunodeficiency virus type 1 (HIV-1) Env (Env375). We developed the following three new simian-human immunodeficiency virus (SHIV) stocks: SHIV-SF162p3S/wild type, SHIV-AE16W, and SHIV-325cH. SHIV-SF162p3S could not be optimized, SHIV-AE16W proved comparable to the parental virus, and SHIV-325cH demonstrated markedly enhanced replicative capacity compared with the parental virus.

The nuclear factor kappa B (NF-B) is a potent transcription factor, activation of which typically results in robust proinflammatory signaling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in the gradual and persistent activation of NF-B, illustrated by proteasome-dependent degradation of the inhibitory NF-B regulator IB and nuclear translocation and phosphorylation of the NF-B subunit p65. PRV-induced persistent activation of NF-B does not result in expression of negative feedback loop genes, like the gene for IBα or A20, and does not trigger expression of prototypical proinflammatory genes, like the gene for tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). In addition, PRV infection inhibits TNF-α-induced canonical NF-B activation. Hence, PRV infection triggers persistent NF-B activation in an unorthodox way and dramatically modulates the NF-B signaling axis, preventing typical proinflammatory gene expression and the responsiveness of cells to canonical NF-B signaling, which may aid the virus in modulating early proinflammatory responses in the infected host.

IMPORTANCE The NF-B transcription factor is activated via different key inflammatory pathways and typically results in the fast expression of several proinflammatory genes as well as negative feedback loop genes to prevent excessive inflammation. In the current report, we describe that infection of cells with the porcine alphaherpesvirus pseudorabies virus (PRV) triggers a gradual and persistent aberrant activation of NF-B, which does not result in expression of hallmark proinflammatory or negative feedback loop genes. In addition, although PRV-induced NF-B activation shares some mechanistic features with canonical NF-B activation, it also shows remarkable differences; e.g., it is largely independent of the canonical IB kinase (IKK) and even renders infected cells resistant to canonical NF-B activation by the inflammatory cytokine TNF-α. Aberrant PRV-induced NF-B activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-B activation.

During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41)₃] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system.

IMPORTANCE The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine.

The entry/fusion complex (EFC) consists of 11 conserved proteins embedded in the membrane envelope of mature poxvirus particles. Poxviruses also encode proteins that localize in cell membranes and negatively regulate superinfection and syncytium formation. The vaccinia virus (VACV) A56/K2 fusion regulatory complex associates with the G9/A16 EFC subcomplex, but functional support for the importance of this interaction was lacking. Here, we describe serially passaging VACV in nonpermissive cells expressing A56/K2 as an unbiased approach to isolate and analyze escape mutants. Viruses forming large plaques in A56/K2 cells increased in successive rounds of infection, indicating the occurrence and enrichment of adaptive mutations. Sequencing of genomes of passaged and cloned viruses revealed mutations near the N terminus of the G9 open reading frame but none in A16 or other genes. The most frequent mutation was His to Tyr at amino acid 44; additional escape mutants had a His-to-Arg mutation at amino acid 44 or a duplication of amino acids 26 to 39. An adaptive Tyr-to-Cys substitution at amino acid 42 was discovered using error-prone PCR to generate additional mutations. Myristoylation of G9 was unaffected by the near-N-terminal mutations. The roles of the G9 mutations in enhancing plaque size were validated by homologous recombination. The mutants exhibited enhanced entry and spread in A56/K2 cells and induced syncytia at neutral pH in HeLa cells despite the expression of A56/K2. The data suggest that the mutations perturb the interaction of G9 with A56/K2, although some association was still detected in detergent-treated infected cell lysates.

IMPORTANCE The entry of enveloped viruses is achieved by the fusion of viral and cellular membranes, a critical step in infection that determines host range and provides targets for vaccines and therapeutics. Poxviruses encode an exceptionally large number of proteins comprising the entry/fusion complex (EFC), which enables infection of diverse cells. Vaccinia virus (VACV), the prototype member of the poxvirus family, also encodes the fusion regulatory proteins A56 and K2, which are displayed on the plasma membrane and may be beneficial by preventing reinfection and cell-cell fusion. Previous studies showed that A56/K2 interacts with the G9/A16 EFC subcomplex in detergent-treated cell extracts. Functional evidence for the importance of this interaction was obtained by serially passaging wild-type VACV in cells that are nonpermissive because of A56/K2 expression. VACV mutants with amino acid substitutions or duplications near the N terminus of G9 were enriched because of their ability to overcome the block to entry imposed by A56/K2.

Disorders of oxygen transport are commonly attributed to inadequate carrying capacity (anemia) but may also relate to inefficient gas exchange by red blood cells (RBCs), a process that is poorly characterized yet assumed to be rapid. Without direct measurements of gas exchange at the single-cell level, the barriers to O2...

Ultrafast spectroscopy is capable of monitoring electronic and vibrational states. For electronic states a few eV apart, an X-ray laser source is required. We propose an alternative method based on the time-domain high-order harmonic spectroscopy where a coherent superposition of the electronic states is first prepared by the strong optical...

One factor that contributes to the high prevalence of fetal alcohol spectrum disorder (FASD) is binge-like consumption of alcohol before pregnancy awareness. It is known that treatments are more effective with early recognition of FASD. Recent advances in retrospective motion correction for the reconstruction of three-dimensional (3D) fetal brain MRI...

HIV-1 persists in cellular reservoirs that can reignite viremia if antiretroviral therapy (ART) is interrupted. Therefore, insight into the nature of those reservoirs may be revealed from the composition of recrudescing viremia following treatment cessation. A minor population of macrophage-tropic (M-tropic) viruses was identified in a library of recombinant viruses...

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in...

Isocitrate dehydrogenase (IDH) mutation is a common genetic abnormality in human malignancies characterized by remarkable metabolic reprogramming. Our present study demonstrated that IDH1-mutated cells showed elevated levels of reactive oxygen species and higher demands on Nrf2-guided glutathione de novo synthesis. Our findings showed that triptolide, a diterpenoid epoxide from Tripterygium...

Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have...

Genetic variants within complement factor H (CFH), a major alternative complement pathway regulator, are associated with the development of age-related macular degeneration (AMD) and other complementopathies. This is explained with the reduced binding of CFH or its splice variant factor H-like protein 1 (FHL-1) to self-ligands or altered self-ligands (e.g.,...

Cellular starvation is typically a consequence of tissue injury that disrupts the local blood supply but can also occur where cell populations outgrow the local vasculature, as observed in solid tumors. Cells react to nutrient deprivation by adapting their metabolism, or, if starvation is prolonged, it can result in cell...

Aerosol–radiation interaction (ARI) plays a significant role in the accumulation of fine particulate matter (PM2.5) by stabilizing the planetary boundary layer and thus deteriorating air quality during haze events. However, modification of photolysis by aerosol scattering or absorbing solar radiation (aerosol–photolysis interaction or API) alters the atmospheric oxidizing capacity, decreases...

Neural networks have become the method of choice in surrogate modeling because of their ability to characterize arbitrary, high-dimensional functions in a data-driven fashion. This paper advocates for the training of surrogates that are 1) consistent with the physical manifold, resulting in physically meaningful predictions, and 2) cyclically consistent with...

G-quadruplex, assembled from a square array of guanine (G) molecules, is an important structure with crucial biological roles in vivo but also a versatile template for ordered functional materials. Although the understanding of G-quadruplex structures is the focus of numerous studies, little is known regarding the control of G-quartet stacking...

The extracellular matrix (ECM) initiates mechanical cues that activate intracellular signaling through matrix–cell interactions. In blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from the ECM and their interaction with...

Recent epidemics demonstrate the global threat of Zika virus (ZIKV), a flavivirus transmitted by mosquitoes. Although infection is usually asymptomatic or mild, newborns of infected mothers can display severe symptoms, including neurodevelopmental abnormalities and microcephaly. Given the large-scale spread, symptom severity, and lack of treatment or prophylaxis, a safe and...

Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire–mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled...

The apparent detection of an exoplanet orbiting Fomalhaut was announced in 2008. However, subsequent observations of Fomalhaut b raised questions about its status: Unlike other exoplanets, it is bright in the optical and nondetected in the infrared, and its orbit appears to cross the debris ring around the star without...

The biological carbon pump (BCP) comprises wide-ranging processes that set carbon supply, consumption, and storage in the oceans’ interior. It is becoming increasingly evident that small changes in the efficiency of the BCP can significantly alter ocean carbon sequestration and, thus, atmospheric CO2 and climate, as well as the functioning...

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the...

Convection is buoyancy-driven flow resulting from unstable density stratification in the presence of a gravitational field. Beyond convection’s central role in myriad engineering heat transfer applications, it underlies many of nature’s dynamical designs on larger-than-human scales. For example, solar heating of Earth’s surface generates buoyancy forces that cause the winds...

Our study in PNAS (1) applies a mathematically based framework to the study of reported emotional experience evoked by music in the United States and China. Germane to theoretical debate, our methods reveal how specific emotions such as “triumph” are more primary across cultures than valence and arousal, a finding...

Cowen et al. (1) leverage modern gains in data science to describe impressive cross-cultural similarities in the perception of musical affect and do so in unprecedented detail. Their approach is innovative and fundamentally empirical. As such, it should have important applications for prediction in the field of affective computing, which...

Proteasomes are essential protease complexes that maintain cellular homeostasis, and aberrant proteasomal activity supports cancer development. The regulatory mechanisms and biological function of the ubiquitin-26S proteasome have been studied extensively, while those of the ubiquitin-independent 20S proteasome system remain obscure. Here, we show that the cap ’n’ collar (CNC) family transcription factor NRF3 specifically enhances 20S proteasome assembly in cancer cells and that 20S proteasomes contribute to colorectal cancer development through ubiquitin-independent proteolysis of the tumor suppressor p53 and retinoblastoma (Rb) proteins. The NRF3 gene is highly expressed in many cancer tissues and cell lines and is important for cancer cell growth. In cancer cells, NRF3 upregulates the assembly of the 20S proteasome by directly inducing the gene expression of the 20S proteasome maturation protein POMP. Interestingly, NRF3 knockdown not only increases p53 and Rb protein levels but also increases p53 activities for tumor suppression, including cell cycle arrest and induction of apoptosis. Furthermore, protein stability and cell viability assays using two distinct proteasome inhibitor anticancer drugs, the 20S proteasome inhibitor bortezomib and the ubiquitin-activating enzyme E1 inhibitor TAK-243, show that the upregulation of the NRF3-POMP axis leads to ubiquitin-independent proteolysis of p53 and Rb and to impaired sensitivity to bortezomib but not TAK-243. More importantly, the NRF3-POMP axis supports tumorigenesis and metastasis, with higher NRF3/POMP expression levels correlating with poor prognoses in patients with colorectal or rectal adenocarcinoma. These results suggest that the NRF3-POMP-20S proteasome assembly axis is significant for cancer development via ubiquitin-independent proteolysis of tumor suppressor proteins.

Recently, abundant evidence has clarified that long noncoding RNAs (lncRNAs) play an oncogenic or anticancer role in the tumorigenesis and development of diverse human cancers. Described as a crucial regulator in some cancers, MIR22HG has not yet been studied in laryngocarcinoma and therefore the underlying regulatory role of MIR22HG in laryngocarcinoma is worth detecting. In this study, MIR22HG expression in laryngocarcinoma cells was confirmed to be downregulated, and upregulated MIR22HG expression led to suppressive effects on laryngocarcinoma cell proliferation and migration. Molecular mechanism assays revealed that MIR22HG sponges miR-5000-3p in laryngocarcinoma cells. Besides, decreased expression of miR-5000-3p suppressed laryngocarcinoma cell proliferation and migration. Moreover, the FBXW7 gene was reported to be a downstream target gene of miR-5000-3p in laryngocarcinoma cells. More importantly, rescue assays verified that FBXW7 depletion or miR-5000-3p upregulation countervailed the repressive effects of MIR22HG overexpression on laryngocarcinoma progression. In addition, E2F6 was proved to be capable of inhibiting MIR22HG transcription in laryngocarcinoma cells. To sum up, E2F6-induced downregulation of MIR22HG promotes laryngocarcinoma progression through the miR-5000-3p/FBXW7 axis.

Microtubule-associated serine/threonine kinase like (MASTL), also known as Greatwall (Gwl) kinase, has an important role in the regulation of mitosis. By inhibiting protein phosphatase 2A (PP2A), it plays a crucial role in activating one of the most important mitotic kinases, known as cyclin-dependent kinase 1 (CDK1). MASTL has been seen to be upregulated in various types of cancers and is also involved in tumor recurrence. It is activated by CDK1 through phosphorylations in the activation/T-loop, but the complete mechanism of its activation is still unclear. Here, we report that AKT phosphorylates MASTL at residue T299, which plays a critical role in its activation. Our results suggest that AKT increases CDK1-mediated phosphorylation and hence the activity of MASTL, which, in turn, promotes mitotic progression through PP2A inhibition. We also show that the oncogenic potential of AKT is augmented by MASTL activation, since AKT-mediated proliferation in colorectal cell lines can be attenuated by inhibiting and/or silencing MASTL. In brief, we report that AKT plays an important role in the progression of mitosis in mammalian cells and that it does so through the phosphorylation and activation of MASTL.

The metabolic state of the brain can greatly impact neurologic function. Evidence of this includes the therapeutic benefit of a ketogenic diet in neurologic diseases, including epilepsy. However, brain lipid bioenergetics remain largely uncharacterized. The existence, capacity, and relevance of mitochondrial fatty acid β-oxidation (FAO) in the brain are highly controversial, with few genetic tools available to evaluate the question. We have provided evidence for the capacity of brain FAO using a pan-brain-specific conditional knockout (KO) mouse incapable of FAO due to the loss of carnitine palmitoyltransferase 2, the product of an obligate gene for FAO (CPT2^B–/–). Loss of central nervous system (CNS) FAO did not result in gross neuroanatomical changes or systemic differences in metabolism. Loss of CPT2 in the brain did not result in robustly impaired behavior. We demonstrate by unbiased and targeted metabolomics that the mammalian brain oxidizes a substantial quantity of long-chain fatty acids in vitro and in vivo. Loss of CNS FAO results in robust accumulation of long-chain acylcarnitines in the brain, suggesting that the mammalian brain mobilizes fatty acids for their oxidation, irrespective of diet or metabolic state. Together, these data demonstrate that the mammalian brain oxidizes fatty acids under normal circumstances with little influence from or on peripheral tissues.

ABSTRACT

The effect of the rapid accumulation of nonsynonymous mutations on the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not yet known. The 3a protein is unique to SARS-CoV and is essential for disease pathogenesis. Our study aimed at determining the nonsynonymous mutations in the 3a protein in SARS-CoV-2 and determining and characterizing the protein’s structure and spatial orientation in comparison to those of 3a in SARS-CoV. A total of 51 different nonsynonymous amino acid substitutions were detected in the 3a proteins among 2,782 SARS-CoV-2 strains. We observed microclonality within the ORF3a gene tree defined by nonsynonymous mutations separating the isolates into distinct subpopulations. We detected and identified six functional domains (I to VI) in the SARS-CoV-2 3a protein. The functional domains were linked to virulence, infectivity, ion channel formation, and virus release. Our study showed the importance of conserved functional domains across the species barrier and revealed the possible role of the 3a protein in the viral life cycle. Observations reported in this study merit experimental confirmation.

IMPORTANCE At the surge of the coronavirus disease 2019 (COVID-19) pandemic, we detected and identified six functional domains (I to VI) in the SARS-CoV-2 3a protein. Our analysis showed that the functional domains were linked to virulence, infectivity, ion channel formation, and virus release in SARS-CoV-2 3a. Our study also revealed the functional importance of conserved domains across the species barrier. Observations reported in this study merit experimental confirmation.

Background:

Patient identification is an important step for advance care planning (ACP) discussions.

Cervical spondylotic myelopathy (CSM) is a neurologic condition that develops insidiously over time as degenerative changes of the spine result in compression of the cord and nearby structures. It is the most common form of spinal cord injury in adults; yet, its diagnosis is often delayed. The purpose of this article is to review the pathophysiology, natural history, diagnosis, and management of CSM with a focus on the recommended timeline for physicians suspecting CSM to refer patients to a spine surgeon. Various processes underlie spondylotic changes of the canal and are separated into static and dynamic factors. Not all patients with evidence of cord compression will present with symptoms, and the progression of disease varies by patient. The hallmark symptoms of CSM include decreased hand dexterity and gait instability as well as sensory and motor dysfunction. magnetic resonance imaging is the imaging modality of choice in patients with suspected CSM, but computed tomography myelography may be used in patients with contraindications. Patients with mild CSM may be treated surgically or nonoperatively, whereas those with moderate-severe disease are treated operatively. Due to the long-term disability that may result from a delay in diagnosis and management, prompt referral to a spine surgeon is recommended for any patient suspected of having CSM. This review provides information and guidelines for practitioners to develop an actionable awareness of CSM.

Purpose:

Clinical evidence shows minimal benefit to vitamin D screening and subsequent treatment in the general population. This study aims to assess the effectiveness of 2 light-touch interventions on reducing vitamin D test orders.

Background:

In 2015, Vyvanse (lisdexamfetamine) became the first Food and Drug Administration (FDA)-approved treatment for binge-eating disorder (BED), a condition first recognized by the DSM–V in 2013. Because pharmaceutical companies use continuing medical education (CME) to help sell drugs, we explored possible bias in CME modules on BED.

Family physician researchers continue to provide assistance to improve family medicine care. Commentaries on social determinants of health lead off this issue. Next, we have several papers on successful interventions by clinicians and/or patients to improve diabetes control, and then other provide information on other practice interventions that make a difference in overall care. Drug advertising continues to mislead. There is costly and nonproductive overuse of specific types of care. Herein is also a Scoping Review of possible indices for determining timely initiation of advance care planning. The issue’s clinical reviews on use of transgender care, cervical myelopathy, and inhaled steroids for chronic obstructive pulmonary disease are pertinent, thorough, and timely.

Kathryn Knight

William Joyce and Tobias Wang

Rafael Dalmau

Gabriela Montejo-Kovacevich, Simon H. Martin, Joana I. Meier, Caroline N. Bacquet, Monica Monllor, Chris D. Jiggins, and Nicola J. Nadeau

Microclimatic variability in tropical forests plays a key role in shaping species distributions and their ability to cope with environmental change, especially for ectotherms. Nonetheless, currently available climatic datasets lack data from the forest interior and, furthermore, our knowledge of thermal tolerance among tropical ectotherms is limited. We therefore studied natural variation in the microclimate experienced by tropical butterflies in the genus Heliconius across their Andean range in a single year. We found that the forest strongly buffers temperature and humidity in the understorey, especially in the lowlands, where temperatures are more extreme. There were systematic differences between our yearly records and macroclimate databases (WorldClim2), with lower interpolated minimum temperatures and maximum temperatures higher than expected. We then assessed thermal tolerance of 10 Heliconius butterfly species in the wild and found that populations at high elevations had significantly lower heat tolerance than those at lower elevations. However, when we reared populations of the widespread H. erato from high and low elevations in a common-garden environment, the difference in heat tolerance across elevations was reduced, indicating plasticity in this trait. Microclimate buffering is not currently captured in publicly available datasets, but could be crucial for enabling upland shifting of species sensitive to heat such as highland Heliconius. Plasticity in thermal tolerance may alleviate the effects of global warming on some widespread ectotherm species, but more research is needed to understand the long-term consequences of plasticity on populations and species.

Ana Gabriela Jimenez, Emily Cornelius Ruhs, Kailey J. Tobin, Katie N. Anderson, Audrey Le Pogam, Lyette Regimbald, and Francois Vezina

Seasonal changes in maximal thermogenic capacity (M_sum) in wild black-capped chickadees suggests that adjustments in metabolic performance are slow and begin to take place before winter peaks. However, when mean minimal ambient temperature (T_a) reaches –10°C, the chickadee phenotype appears to provide enough spare capacity to endure days with colder T_a, down to –20°C or below. This suggests that birds could also maintain a higher antioxidant capacity as part of their cold-acclimated phenotype to deal with sudden decreases in temperature. Here, we tested how environmental mismatch affected oxidative stress by comparing cold-acclimated (–5°C) and transition (20°C) phenotypes in chickadees exposed to an acute 15°C drop in temperature with that of control individuals. We measured superoxide dismutase, catalase and glutathione peroxidase activities, as well as lipid peroxidation damage and antioxidant scavenging capacity in pectoralis muscle, brain, intestine and liver. We generally found differences between seasonal phenotypes and across tissues, but no differences with respect to an acute cold drop treatment. Our data suggest oxidative stress is closely matched to whole-animal physiology in cold-acclimated birds compared with transition birds, implying that changes to the oxidative stress system happen slowly.

Irene Verhagen, Barbara M. Tomotani, Phillip Gienapp, and Marcel E. Visser

Phenotypic plasticity is an important mechanism by which an individual can adapt its seasonal timing to predictable, short-term environmental changes by using predictive cues. Identification of these cues is crucial to forecast the response of species to long-term environmental change and to study their potential to adapt. Individual great tits (Parus major) start reproduction early under warmer conditions in the wild, but whether this effect is causal is not well known. We housed 36 pairs of great tits in climate-controlled aviaries and 40 pairs in outdoor aviaries, where they bred under artificial contrasting temperature treatments or in semi-natural conditions, respectively, for two consecutive years, using birds from lines selected for early and late egg laying. We thus obtained laying dates in two different thermal environments for each female. Females bred earlier under warmer conditions in climate-controlled aviaries, but not in outdoor aviaries. The latter was inconsistent with laying dates from our wild population. Further, early selection line females initiated egg laying consistently ~9 days earlier than late selection line females in outdoor aviaries, but we found no difference in the degree of plasticity (i.e. the sensitivity to temperature) in laying date between selection lines. Because we found that temperature causally affects laying date, climate change will lead to earlier laying. This advancement is, however, unlikely to be sufficient, thereby leading to selection for earlier laying. Our results suggest that natural selection may lead to a change in mean phenotype, but not to a change in the sensitivity of laying dates to temperature.

Ewa Komar, Dina K. N. Dechmann, Nicolas J. Fasel, Marcin Zegarek, and Ireneusz Ruczynski

Balancing energy budgets can be challenging, especially in periods of food shortage, adverse weather conditions and increased energy demand due to reproduction. Bats have particularly high energy demands compared to other mammals and regularly use torpor to save energy. However, while torpor limits energy expenditure, it can also downregulate important processes, such as sperm production. This constraint could result in a trade-off between energy saving and future reproductive capacity. We mimicked harsh conditions by restricting food and tested the effect on changes in body mass, torpor use and seasonal sexual maturation in male parti-coloured bats (Vespertilio murinus). Food-restricted individuals managed to maintain their initial body mass, while in well-fed males, mass increased. Interestingly, despite large differences in food availability, there were only small differences in torpor patterns. However, well-fed males reached sexual maturity up to half a month earlier. Our results thus reveal a complex trade-off in resource allocation; independent of resource availability, males maintain a similar thermoregulation strategy and favour fast sexual maturation, but limited resources and low body mass moderate this latter process.