Title: Too Much or Too Little Weight May Worsen Rheumatoid Arthritis
Category: Health News
Created: 4/30/2018 12:00:00 AM
Last Editorial Review: 4/30/2018 12:00:00 AM

Title: 'What's That Word?' Fitness Helps Seniors Find It
Category: Health News
Created: 4/30/2018 12:00:00 AM
Last Editorial Review: 5/1/2018 12:00:00 AM

Title: Sex Still Matters to Many Seniors, Survey Finds
Category: Health News
Created: 5/3/2018 12:00:00 AM
Last Editorial Review: 5/4/2018 12:00:00 AM

Title: As Sense of Smell Fades, Does Death Come Closer?
Category: Health News
Created: 4/29/2019 12:00:00 AM
Last Editorial Review: 4/30/2019 12:00:00 AM

Title: Morning Exercise Kick-Starts Seniors' Brains
Category: Health News
Created: 5/2/2019 12:00:00 AM
Last Editorial Review: 5/2/2019 12:00:00 AM

Title: Health Tip: Sensitive Teeth
Category: Health News
Created: 5/2/2019 12:00:00 AM
Last Editorial Review: 5/2/2019 12:00:00 AM

Title: As Finals Draw Near, College Kids' Diets Worsen
Category: Health News
Created: 5/3/2019 12:00:00 AM
Last Editorial Review: 5/3/2019 12:00:00 AM

Title: AHA News: Essential Advice for Stressed-Out Essential Workers
Category: Health News
Created: 4/24/2020 12:00:00 AM
Last Editorial Review: 4/27/2020 12:00:00 AM

Title: More Airlines to Make Passenger Face Coverings Mandatory
Category: Health News
Created: 5/1/2020 12:00:00 AM
Last Editorial Review: 5/1/2020 12:00:00 AM

Title: Many Seniors Think They See Better Than They Actually Do
Category: Health News
Created: 2/5/2020 12:00:00 AM
Last Editorial Review: 2/6/2020 12:00:00 AM

NCBI has developed a new web presentation style called PubReader, which offers you an easier way to use your Web browser to read the articles in PMC. Designed particularly for enhancing readability and navigation on tablet and other small screen devices, PubReader can also be used on desktops and laptops and from multiple web browsers. For more information on PubReader, see the article in the November-December issue of the NLM Technical Bulletin.

Title: When Is Surgery Not Safe for Seniors?
Category: Health News
Created: 3/12/2020 12:00:00 AM
Last Editorial Review: 3/13/2020 12:00:00 AM

Title: Pneumonia More Deadly Than Hip Fractures for Hospitalized Seniors
Category: Health News
Created: 4/23/2020 12:00:00 AM
Last Editorial Review: 4/24/2020 12:00:00 AM

Title: Vitamin D Might Aid Seniors' Recovery From Hip Fracture: Study
Category: Health News
Created: 4/2/2020 12:00:00 AM
Last Editorial Review: 4/3/2020 12:00:00 AM

Title: Crafting With Dry Pasta, Play-Doh Safe for Kids With Gluten Sensitivity: Study
Category: Health News
Created: 2/4/2020 12:00:00 AM
Last Editorial Review: 2/5/2020 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: Many Seniors Leave Hospital With New Disabilities
Category: Health News
Created: 3/2/2020 12:00:00 AM
Last Editorial Review: 3/3/2020 12:00:00 AM

Title: Sleepy Seniors Have Higher Health Risks
Category: Health News
Created: 3/2/2020 12:00:00 AM
Last Editorial Review: 3/3/2020 12:00:00 AM

Title: Helping Seniors Manage Meds After Hospital Reduces Readmission: Study
Category: Health News
Created: 3/3/2020 12:00:00 AM
Last Editorial Review: 3/4/2020 12:00:00 AM

Title: Seniors, Getting Off the Sofa Brings Big Health Benefits
Category: Health News
Created: 3/5/2020 12:00:00 AM
Last Editorial Review: 3/6/2020 12:00:00 AM

Title: How Pets Can Be True Lifesavers for Seniors
Category: Health News
Created: 4/3/2020 12:00:00 AM
Last Editorial Review: 4/3/2020 12:00:00 AM

Title: Back in Touch: Technology Restores Hand Sensitivity to Young Quadraplegic
Category: Health News
Created: 4/23/2020 12:00:00 AM
Last Editorial Review: 4/24/2020 12:00:00 AM

Title: How Long Does a Laparoscopic Nissen Fundoplication Last?
Category: Procedures and Tests
Created: 4/29/2020 12:00:00 AM
Last Editorial Review: 4/29/2020 12:00:00 AM

The invertebrate trace fossils Selenichnites rossendalensis and Crescentichnus tesiltus are recorded and described from the Middle Jurassic Gristhorpe Member of the Cloughton Formation of the Cleveland Basin. This is the first record of these ichnospecies from the basin and now completes the occurrence of these and other traces assumed to have been made by limulids from all three non-marine formations of the Ravenscar Group.

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

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

Merkel cell polyomavirus (MCPyV) is the only polyomavirus known to be associated with tumorigenesis in humans. Similarly to other polyomaviruses, MCPyV expresses a large tumor antigen (LT-Ag) that, together with a small tumor antigen (sT-Ag), contributes to cellular transformation and that is of critical importance for the initiation of the viral DNA replication. Understanding the cellular protein network regulated by MCPyV early proteins will significantly contribute to our understanding of the natural MCPyV life cycle as well as of the mechanisms by which the virus contributes to cellular transformation. We here describe KRAB-associated protein 1 (Kap1), a chromatin remodeling factor involved in cotranscriptional regulation, as a novel protein interaction partner of MCPyV T antigens sT and LT. Kap1 knockout results in a significant increase in the level of viral DNA replication that is highly suggestive of Kap1 being an important host restriction factor during MCPyV infection. Differently from other DNA viruses, MCPyV gene expression is unaffected in the absence of Kap1 and Kap1 does not associate with the viral genome. Instead, we show that in primary normal human dermal fibroblast (nHDF) cells, MCPyV DNA replication, but not T antigen expression alone, induces ataxia telangiectasia mutated (ATM) kinase-dependent Kap1 S824 phosphorylation, a mechanism that typically facilitates repair of double-strand breaks in heterochromatin by arresting the cells in G₂. We show that MCPyV-induced inhibition of cell proliferation is mainly conferred by residues within the origin binding domain and thereby by viral DNA replication. Our data suggest that phosphorylation of Kap1 and subsequent Kap1-dependent G₂ arrest/senescence represent host defense mechanisms against MCPyV replication in nHDF cells.

IMPORTANCE We here describe Kap1 as a restriction factor in MCPyV infection. We report a novel, indirect mechanism by which Kap1 affects MCPyV replication. In contrast with from other DNA viruses, Kap1 does not associate with the viral genome in MCPyV infection and has no impact on viral gene expression. In MCPyV-infected nHDF cells, Kap1 phosphorylation (pKap1 S824) accumulates because of genomic stress mainly induced by viral DNA replication. In contrast, ectopic expression of LT or LT MCPyV mutants, previously shown to be important for induction of genotoxic stress, does not result in a similar extent of pKap1 accumulation. We show that cells actively replicating MCPyV accumulate pKap1 (in a manner dependent on the presence of ATM) and display a senescence phenotype reflected by G₂ arrest. These results are supported by transcriptome analyses showing that LT antigen, in a manner dependent on the presence of Kap1, induces expression of secreted factors, which is known as the senescence-associated secretory phenotype (SASP).

ABSTRACT

Recent global advocacy efforts have highlighted the importance of development of a vaccine against group A Streptococcus (GAS). Combo5 is a non-M protein-based vaccine that provides protection against GAS skin infection in mice and reduces the severity of pharyngitis in nonhuman primates. However, Combo5 with the addition of aluminum hydroxide (alum) as an adjuvant failed to protect against invasive GAS infection of mice. Here, we show that formulation of Combo5 with adjuvants containing saponin QS21 significantly improves protective efficacy, even though all 7 adjuvants tested generated high antigen-specific IgG antibody titers, including alum. Detailed characterization of Combo5 formulated with SMQ adjuvant, a squalene-in-water emulsion containing a TLR4 agonist and QS21, showed significant differences from the results obtained with alum in IgG subclasses generated following immunization, with an absence of GAS opsonizing antibodies. SMQ, but not alum, generated strong interleukin-6 (IL-6), gamma interferon (IFN-), and tumor necrosis alpha (TNF-α) responses. This work highlights the importance of adjuvant selection for non-M protein-based GAS vaccines to optimize immune responses and protective efficacy.

IMPORTANCE Availability of a group A Streptococcus vaccine remains an unmet public health need. Here, we tested different adjuvant formulations to improve the protective efficacy of non-M protein vaccine Combo5 in an invasive disease model. We show that novel adjuvants can dramatically shape the type of immune response developed following immunization with Combo5 and significantly improve protection. In addition, protection afforded by Combo5 is not mediated by opsonizing antibodies, believed to be the main correlate of protection against GAS infections. Overall, this report highlights the importance of adjuvant selection in raising protective immune responses against GAS invasive infection. Adjuvants that can provide a more balanced Th1/Th2-type response may be required to optimize protection of GAS vaccines, particularly those based on non-M protein antigens.

ABSTRACT

The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp⁰] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase.

IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.

ABSTRACT

Bacterial flagellar motility plays an important role in many processes that occur at surfaces or in hydrogels, including adhesion, biofilm formation, and bacterium-host interactions. Consequently, expression of flagellar genes, as well as genes involved in biofilm formation and virulence, can be regulated by the surface contact. In a few bacterial species, flagella themselves are known to serve as mechanosensors, where an increased load on flagella experienced during surface contact or swimming in viscous media controls gene expression. In this study, we show that gene regulation by motility-dependent mechanosensing is common among pathogenic Escherichia coli strains. This regulatory mechanism requires flagellar rotation, and it enables pathogenic E. coli to repress flagellar genes at low loads in liquid culture, while activating motility in porous medium (soft agar) or upon surface contact. It also controls several other cellular functions, including metabolism and signaling. The mechanosensing response in pathogenic E. coli depends on the negative regulator of motility, RflP (YdiV), which inhibits basal expression of flagellar genes in liquid. While no conditional inhibition of flagellar gene expression in liquid and therefore no upregulation in porous medium was observed in the wild-type commensal or laboratory strains of E. coli, mechanosensitive regulation could be recovered by overexpression of RflP in the laboratory strain. We hypothesize that this conditional activation of flagellar genes in pathogenic E. coli reflects adaptation to the dual role played by flagella and motility during infection.

IMPORTANCE Flagella and motility are widespread virulence factors among pathogenic bacteria. Motility enhances the initial host colonization, but the flagellum is a major antigen targeted by the host immune system. Here, we demonstrate that pathogenic E. coli strains employ a mechanosensory function of the flagellar motor to activate flagellar expression under high loads, while repressing it in liquid culture. We hypothesize that this mechanism allows pathogenic E. coli to regulate its motility dependent on the stage of infection, activating flagellar expression upon initial contact with the host epithelium, when motility is beneficial, but reducing it within the host to delay the immune response.

ABSTRACT

Streptococcus pneumoniae (or pneumococcus) is a highly prevalent human pathogen. Toll-like receptors (TLRs) function as immune sensors that can trigger host defenses against this bacterium. Defects in TLR-activated signaling pathways, including deficiency in the adaptor protein myeloid differentiation factor 88 (MyD88), are associated with markedly increased susceptibility to infection. However, the individual MyD88-dependent TLRs predominantly involved in antipneumococcal defenses have not been identified yet. Here we find that triple knockout mice simultaneously lacking TLR7, TLR9, and TLR13, which sense the presence of bacterial DNA (TLR9) and RNA (TLR7 and TLR13) in the phagolysosomes of phagocytic cells, display a phenotype that largely resembles that of MyD88-deficient mice and rapidly succumb to pneumococcal pneumonitis due to defective neutrophil influx into the lung. Accordingly, TLR7/9/13 triple knockout resident alveolar macrophages were largely unable to respond to pneumococci with the production of neutrophil-attracting chemokines and cytokines. Mice with single deficiencies of TLR7, TLR9, or TLR13 showed unaltered ability to control lung infection but were moderately more susceptible to encephalitis, in association with a decreased ability of microglia to mount cytokine responses in vitro. Our data point to a dominant, tissue-specific role of nucleic acid-sensing pathways in innate immune recognition of S. pneumoniae and also show that endosomal TLRs are largely capable of compensating for the absence of each other, which seems crucial to prevent pneumococci from escaping immune recognition. These results may be useful to develop novel strategies to treat infections by antibiotic-resistant pneumococci based on stimulation of the innate immune system.

IMPORTANCE The pneumococcus is a bacterium that frequently causes infections in the lungs, ears, sinus cavities, and meninges. During these infections, body defenses are triggered by tissue-resident cells that use specialized receptors, such as Toll-like receptors (TLRs), to sense the presence of bacteria. We show here that pneumococci are predominantly detected by TLRs that are located inside intracellular vacuoles, including endosomes, where these receptors can sense the presence of nucleic acids released from ingested bacteria. Mice that simultaneously lacked three of these receptors (specifically, TLR7, TLR9, and TLR13) were extremely susceptible to lung infection and rapidly died after inhalation of pneumococci. Moreover, tissue-resident macrophages from these mice were impaired in their ability to respond to the presence of pneumococci by producing inflammatory mediators capable of recruiting polymorphonuclear leucocytes to infection sites. This information may be useful to develop drugs to treat pneumococcal infections, particularly those caused by antibiotic-resistant strains.

ABSTRACT

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often rewired such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this rewired background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations as PAO1, and in E90, RhlR plays a significant role in mediating cytotoxicity in a three-dimensional lung epithelium cell model. These data illuminate a rewired LasR-independent RhlR regulon in chronic infection isolates and suggest further investigation of RhlR as a possible target for therapeutic development in chronic infections.

IMPORTANCE Pseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Many isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR in chronic P. aeruginosa infections.

ABSTRACT

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.

IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

ABSTRACT

Channelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. "Bacteriorhodopsin-like" cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, the sequences of BCCR match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here, we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption, Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function.

IMPORTANCE Cation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-visible (UV-vis) spectroscopy and photoelectrophysiology reveal that the desensitization is caused by rapid accumulation of a nonconductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

To the Editor—The North Carolina Medical Board and North Carolina Medical Society have concerns regarding the Correspondence To the Editor in the March/April 2019 issue of the North Carolina Medical Journal titled, "Aid in Dying in North Carolina" [1]. Although we recognize the beliefs shared by the individual authors were not intended to be conclusive guidance regarding the status of aid in dying (AID) in North Carolina, we feel compelled to respond with a few clarifying notes.

The authors of the correspondence opine: "In light of the legal analysis of North Carolina law, we feel confident that AID can be provided to patients who request it" and that "physicians can provide AID ... without risk of a viable criminal or disciplinary action" [1].

In all matters of medical practice, including end-of-life matters, physicians and physician assistants must meet the standards of acceptable and prevailing medical practice and the ethics of the medical profession. If the Medical Board receives a complaint related to AID, it will evaluate the complaint and determine, utilizing expert consultants, whether the physician engaged in unprofessional conduct as defined by the North Carolina Medical Practice Act.

Further, disagreement exists within the medical community regarding the role of clinicians in medical AID. In one national survey, there was no consensus about the acceptability of AID among physicians and other health care professionals caring for older adults [2]. Respondents also expressed concerns about AID applied to vulnerable populations, including those with low health literacy, low English proficiency, disability, dependency,...

Objective

To investigate the effect of somatic, postzygotic, gain-of-function mutation of Endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) encoding hypoxia-inducible factor-2α (HIF-2α) on posterior fossa development and spinal dysraphism in EPAS1 gain-of-function syndrome, which consists of multiple paragangliomas, somatostatinoma, and polycythemia.

Calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK)-mediated calcium signaling has been widely reported to function in plant development and various stress responses, particularly in ion homeostasis. Sugars are the most important primary metabolites, and thus sugar homeostasis requires precise regulation. Here, we describe a CBL2-CIPK6-Tonoplast-Localized Sugar Transporter2 (TST2) molecular module in cotton (Gossypium hirsutum) that regulates plant sugar homeostasis, in particular Glc homeostasis. GhCIPK6 is recruited to the tonoplast by GhCBL2 and interacts with the tonoplast-localized sugar transporter GhTST2. Overexpression of either GhCBL2, GhCIPK6, or GhTST2 was sufficient to promote sugar accumulation in transgenic cotton, whereas RNAi-mediated knockdown of GhCIPK6 expression or CRISPR-Cas9-mediated knockout of GhTST2 resulted in significantly decreased Glc content. Moreover, mutation of GhCBL2 or GhTST2 in GhCIPK6-overexpressing cotton reinstated sugar contents comparable to wild-type plants. Heterologous expression of GhCIPK6 in Arabidopsis (Arabidopsis thaliana) also promoted Glc accumulation, whereas mutation of AtTST1/2 in GhCIPK6-overexpressing Arabidopsis similarly reinstated wild-type sugar contents, thus indicating conservation of CBL2-CIPK6-TST2-mediated sugar homeostasis among different plant species. Our characterization of the molecular players behind plant sugar homeostasis may be exploited to improve sugar contents and abiotic stress resistance in plants.

Terpene volatiles are found in many important fruit crops, but their relationship to flavor is poorly understood. Here, we demonstrate using sensory descriptive and discriminant analysis that 1,8-cineole contributes a key floral/eucalyptus note to the aroma of ripe 'Hort16A’ kiwifruit (Actinidia chinensis). Two quantitative trait loci (QTLs) for 1,8-cineole production were identified on linkage groups 27 and 29a in a segregating A. chinensis population, with the QTL on LG29a colocating with a complex cluster of putative terpene synthase (TPS)-encoding genes. Transient expression in Nicotiana benthamiana and analysis of recombinant proteins expressed in Escherichia coli showed four genes in the cluster (AcTPS1a–AcTPS1d) encoded functional TPS enzymes, which produced predominantly sabinene, 1,8-cineole, geraniol, and springene, respectively. The terpene profile produced by AcTPS1b closely resembled the terpenes detected in red-fleshed A. chinensis. AcTPS1b expression correlated with 1,8-cineole content in developing/ripening fruit and also showed a positive correlation with 1,8-cineole content in the mapping population, indicating the basis for segregation is an expression QTL. Transient overexpression of AcTPS1b in Actinidia eriantha fruit confirmed this gene produced 1,8-cineole in Actinidia. Structure-function analysis showed AcTPS1a and AcTPS1b are natural variants at key TPS catalytic site residues previously shown to change enzyme specificity in vitro. Together, our results indicate that AcTPS1b is a key gene for production of the signature flavor terpene 1,8-cineole in ripe kiwifruit. Using a sensory-directed strategy for compound identification provides a rational approach for applying marker-aided selection to improving flavor in kiwifruit as well as other fruits.

Purpose of review

Diverse adverse events have been associated with administration of glatiramer acetate (GA), mainly local reactions at the injection site. Other, less frequent generalized reactions include isolated postinjection reactions and anaphylaxis, which may lead to discontinuation of GA.

LuxS/AI-2 is an important quorum sensing system which affects the growth, biofilm formation, virulence, and metabolism of bacteria. LuxS is encoded by the luxS gene, but how this gene is associated with a diverse array of physiological activities in Edwardsiella piscicida (E. piscicida) is not known. Here, we constructed an luxS gene mutant strain, the luxS strain, to identify how LuxS/AI-2 affects pathogenicity. The results showed that LuxS was not found in the luxS gene mutant strain, and this gene deletion decreased E. piscicida growth compared to that of the wild-type strain. Meanwhile, the wild-type strain significantly increased penetration and motility in mucin compared to levels with the luxS strain. The 50% lethal dose (LD₅₀) of the E. piscicida luxS strain for zebrafish was significantly higher than that of the wild-type strain, which suggested that the luxS gene deletion could attenuate the strain’s virulence. The AI-2 activities of EIB202 were 56-fold higher than those in the luxS strain, suggesting that the luxS gene promotes AI-2 production. Transcriptome results demonstrated that between cells infected with the luxS strain and those infected with the wild-type strain 46 genes were significantly differentially regulated, which included 34 upregulated genes and 12 downregulated genes. Among these genes, the largest number were closely related to cell immunity and signaling systems. In addition, the biofilm formation ability of EIB202 was significantly higher than that of the luxS strain. The supernatant of EIB202 increased the biofilm formation ability of the luxS strain, which suggested that the luxS gene and its product LuxS enhanced biofilm formation in E. piscicida. All results indicate that the LuxS/AI-2 quorum sensing system in E. piscicida promotes its pathogenicity through increasing a diverse array of physiological activities.

Antimicrobial peptides play an important role in host defense against Vibrio cholerae. Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS, regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carR^r and carR^s alleles, respectively, and replaced the carR^s allele of a sensitive strain with the carR^r allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarR^s protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR-regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca²⁺ but increased with a rise in pH. The downregulation of almEFG in CarR^s strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains.

Although pain is a prevalent nonmotor symptom in Parkinson’s disease (PD), it is undertreated, in part because of our limited understanding of the underlying mechanisms. Considering that the basal ganglia are implicated in pain sensation, and that their synaptic outputs are controlled by the subthalamic nucleus (STN), we hypothesized that...

Peter J. Edmunds and Scott C. Burgess

Experiments with coral fragments (i.e. nubbins) have shown that net calcification is depressed by elevated P_CO2. Evaluating the implications of this finding requires scaling of results from nubbins to colonies, yet the experiments to codify this process have not been carried out. Building from our previous research demonstrating that net calcification of Pocillopora verrucosa (2–13 cm diameter) was unaffected by P_CO2 (400 and 1000 µatm) and temperature (26.5 and 29.7°C), we sought generality to this outcome by testing how colony size modulates P_CO2 and temperature sensitivity in a branching acroporid. Together, these taxa represent two of the dominant lineages of branching corals on Indo-Pacific coral reefs. Two trials conducted over 2 years tested the hypothesis that the seasonal range in seawater temperature (26.5 and 29.2°C) and a future P_CO2 (1062 µatm versus an ambient level of 461 µatm) affect net calcification of an ecologically relevant size range (5–20 cm diameter) of colonies of Acropora hyacinthus. As for P. verrucosa, the effects of temperature and P_CO2 on net calcification (mg day^–1) of A. verrucosa were not statistically detectable. These results support the generality of a null outcome on net calcification of exposing intact colonies of branching corals to environmental conditions contrasting seasonal variation in temperature and predicted future variation in P_CO2. While there is a need to expand beyond an experimental culture relying on coral nubbins as tractable replicates, rigorously responding to this need poses substantial ethical and logistical challenges.

Surendra Singh Patel, Sanyami Zunjarrao, and Beena Pillai

Eisenia fetida, the common vermicomposting earthworm, shows robust regeneration of posterior segments removed by amputation. During the period of regeneration, the newly formed tissue initially contains only undifferentiated cells but subsequently differentiates into a variety of cell types including muscle, nerve and vasculature. Transcriptomics analysis, reported previously, provided a number of candidate non-coding RNAs that were induced during regeneration. We found that one such long non-coding RNA (lncRNA) is expressed in the skin, only at the base of newly formed chaetae. The spatial organization and precise arrangement of the regenerating chaetae and the cells expressing the lncRNA on the ventral side clearly support a model wherein the regenerating tissue contains a zone of growth and cell division at the tip and a zone of differentiation at the site of amputation. The temporal expression pattern of the lncRNA, named Neev, closely resembled the pattern of chitin synthase genes, implicated in chaetae formation. We found that the lncRNA has 49 sites for binding a set of four microRNAs (miRNAs) while the chitin synthase 8 mRNA has 478 sites. The over-representation of shared miRNA sites suggests that lncRNA Neev may act as a miRNA sponge to transiently de-repress chitin synthase 8 during formation of new chaetae in the regenerating segments of Eisenia fetida.

Karen L. Carleton, Daniel Escobar-Camacho, Sara M. Stieb, Fabio Cortesi, and N. Justin Marshall

Among vertebrates, teleost eye diversity exceeds that found in all other groups. Their spectral sensitivities range from ultraviolet to red, and the number of visual pigments varies from 1 to over 40. This variation is correlated with the different ecologies and life histories of fish species, including their variable aquatic habitats: murky lakes, clear oceans, deep seas and turbulent rivers. These ecotopes often change with the season, but fish may also migrate between ecotopes diurnally, seasonally or ontogenetically. To survive in these variable light habitats, fish visual systems have evolved a suite of mechanisms that modulate spectral sensitivities on a range of timescales. These mechanisms include: (1) optical media that filter light, (2) variations in photoreceptor type and size to vary absorbance and sensitivity, and (3) changes in photoreceptor visual pigments to optimize peak sensitivity. The visual pigment changes can result from changes in chromophore or changes to the opsin. Opsin variation results from changes in opsin sequence, opsin expression or co-expression, and opsin gene duplications and losses. Here, we review visual diversity in a number of teleost groups where the structural and molecular mechanisms underlying their spectral sensitivities have been relatively well determined. Although we document considerable variability, this alone does not imply functional difference per se. We therefore highlight the need for more studies that examine species with known sensitivity differences, emphasizing behavioral experiments to test whether such differences actually matter in the execution of visual tasks that are relevant to the fish.

Annette E. Allen, Joshua W. Mouland, Jessica Rodgers, Beatriz Bano-Otalora, Ronald H. Douglas, Glen Jeffery, Anthony A. Vugler, Timothy M. Brown, and Robert J. Lucas

An animal's temporal niche – the time of day at which it is active – is known to drive a variety of adaptations in the visual system. This includes variations in the topography, spectral sensitivity and density of retinal photoreceptors, and changes in the eye's gross anatomy and spectral transmission characteristics. We have characterised visual spectral sensitivity in the murid rodent Rhabdomys pumilio (‘the four-striped grass mouse’), which is the same family as (nocturnal) mice and rats, but exhibits a strong diurnal niche. As is common in diurnal species, the Rhabdomys lens acts as a long-pass spectral filter, providing limited transmission of light <400nm. Conversely, we found strong sequence homologies with the Rhabdomys SWS and MWS opsins and those of related nocturnal species (mice and rats) whose SWS opsins are maximally sensitive in the near UV. We continued to assess in vivo spectral sensitivity of cone vision using electroretinography and multi-channel recordings from the visual thalamus. These revealed that responses across the human visible range could be adequately described by those of a single pigment (assumed to be MWS opsin) maximally sensitive ~500nm, but that sensitivity in the near UV required inclusion of a second pigment whose peak sensitivity lay well into the UV range (_max <400nm, likely ~360nm). We therefore conclude that, despite the UV-filtering effects of the lens, the Rhabdomys retains an SWS pigment with a UV-A _max. In effect, this somewhat paradoxical combination of long-pass lens and UV-A _max results in narrow-band sensitivity for SWS cone pathways in the UV-A range.

Jessica M. Judson, Dawn M. Reding, and Anne M. Bronikowski

Immunosenescence is a well-known phenomenon in mammal systems, but its relevance in other long-lived vertebrates is less understood. Further, the influence of age and reproductive effort on immune function in long-lived species can be challenging to assess, as long-term data are scarce and it is often difficult to sample the oldest age classes. We used the painted turtle (Chrysemys picta) to test hypotheses of immunosenescence and a trade-off between reproductive output and immune function in a population of a long-lived vertebrate that has been monitored for over 30 years. These long-term data are utilized to employ a unique approach of aging turtles with mark-recapture data and population-specific growth modeling to obtain more accurate estimates of age. We analyzed natural antibodies, lysis ability, and bactericidal competence in 126 individuals from 1 to 33 years of age captured during May and June in 2011. Older turtles exhibited greater natural antibody levels than young individuals across sexes. Young females with large clutches exhibited greater lysis ability, while older females with large clutches had decreased lysis ability, suggesting a trade-off between reproductive output and immune function conditional upon age. However, bactericidal competence increased later in the nesting season for older females. Our study rejects the hypothesis of immunosenescence in a long-lived turtle, despite evidence of actuarial and reproductive senescence in this population. Additionally, we detected mixed evidence for a trade-off between reproduction and immune health.