β-lactam antibiotics are the most commonly used antibacterial agents and growing resistance to these drugs is a concern. Metallo-β-lactamases are a diverse set of enzymes that catalyze the hydrolysis of a broad range of β-lactam drugs including carbapenems. This diversity is reflected in the observation that the enzyme mechanisms differ based on whether one or two zincs are bound in the active site which, in turn, is dependent on the subclass of β-lactamase. The dissemination of the genes encoding these enzymes among Gram-negative bacteria has made them an important cause of resistance. In addition, there are currently no clinically available inhibitors to block metallo-β-lactamase action. This review summarizes the numerous studies that have yielded insights into the structure, function, and mechanism of action of these enzymes.
β-lactamase; antibiotic resistance; carbapenem; zinc metallo-enzyme
Antimuscle specific kinase (anti-MuSK) myasthenia (AMM) differs from antiacetylcholine receptor myasthenia gravis in exhibiting more focal muscle involvement (neck, shoulder, facial, and bulbar muscles) with wasting of the involved, primarily axial, muscles. AMM is not associated with thymic hyperplasia and responds poorly to anticholinesterase treatment. Animal models of AMM have been induced in rabbits, mice, and rats by immunization with purified xenogeneic MuSK ectodomain, and by passive transfer of large quantities of purified serum IgG from AMM patients into mice. The models have confirmed the pathogenic role of the MuSK antibodies in AMM and have demonstrated the involvement of both the presynaptic and postsynaptic components of the neuromuscular junction. The observations in this human disease and its animal models demonstrate the role of MuSK not only in the formation of this synapse but also in its maintenance.
animal models; autoimmune; muscle-specific kinase; muscle wasting; MuSK; myasthenia; neuromuscular junction; synapse
Unlike their protein “roommates” and their nucleic acid “cousins,” carbohydrates remain an enigmatic arm of biology. The central reason for the difficulty in fully understanding how carbohydrate structure and biological function are tied is the nontemplate nature of their synthesis and the resulting heterogeneity. The goal of this collection of expert reviews is to highlight what is known about how carbohydrates and their binding partners—the microbial (non-self), tumor (altered-self), and host (self)—cooperate within the immune system, while also identifying areas of opportunity to those willing to take up the challenge of understanding more about how carbohydrates influence immune responses. In the end, these reviews will serve as specific examples of how carbohydrates are as integral to biology as are proteins, nucleic acids, and lipids. Here, we attempt to summarize general concepts on glycans and glycan-binding proteins (mainly C-type lectins, siglecs, and galectins) and their contributions to the biology of immune responses in physiologic and pathologic settings.
glycobiology; glycoimmunology; glycans; lectins; C-type lectins; siglecs; galectins
Many Gram-negative and Gram-positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many—and quite possibly all—bacteria, the peptidoglycan fragments are recovered and recycled. While cell-wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell-wall–targeting antibiotics and to regulate resistance mechanisms. In several Gram-negative pathogens, anhydro-MurNAc-peptide cell-wall fragments regulate AmpC β-lactamase induction. In some Gram-positive organisms, short peptides derived from the cell wall regulate the induction of both β-lactamase and β-lactam-resistant penicillin-binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell-wall-targeted antibiotics. Indeed, such inhibitors improve the potency of β-lactams in vitro against inducible AmpC β-lactamase-producing bacteria. We describe the key steps of cell-wall remodeling and recycling, the regulation of resistance mechanisms by cell-wall recycling, and recent advances toward the discovery of cell-wall recycling inhibitors.
AmpD; AmpG; AmpR; autolysin; BlaR1; Escherichia coli; lytic transglycosylase; MRSA; NagZ; PBP2a; Staphylococcus aureus
The bactericidal, cell membrane-targeting lipopeptide antibiotic daptomycin (DAP) is an important agent in treating invasive Staphylococcus aureus infections. However, there have been numerous recent reports of development of daptomycin-resistance (DAP-R) during therapy with this agent. The mechanisms of DAP-R in S. aureus appear to be quite diverse. DAP-R strains often exhibit progressive accumulation of single nucleotide polymorphisms in the multipeptide resistance factor gene (mprF) and the yycFG components of the yycFGHI operon. Both loci are involved in key cell membrane (CM) events, with mprF being responsible for the synthesis and outer CM translocation of the positively-charged phospholipid, lysyl-phosphotidylglycerol (L-PG), while the yyc operon is involved in the generalized response to stressors such as antimicrobials. In addition, other perturbations of the CM have been identified in DAP-R strains including: extremes in CM order; resistance to CM depolarization and permeabilization; and reduced surface binding of DAP. Moreover, modifications of the cell wall (CW) appear to also contribute to DAP-R, including enhanced expression of the dlt operon (involved in D-alanylation of CW teichoic acids) and progressive CW thickening.
Staphylococcus aureus; daptomycin; antibiotic resistance; endocarditis
The development of invasive aspergillosis is a feared complication for immunocompromised patients. Despite the use of antifungal agents with excellent bioactivity, the morbidity and mortality rate for invasive aspergillosis remains unacceptably high. Defects within the innate immune response portend the highest risk for patients, but detailed knowledge of molecular pathways in neutrophils and macrophages in response to this fungal pathogen is lacking. Phagocytosis of fungal spores is a key step that places the pathogen into a phagosome, a membrane-delimited compartment that undergoes maturation and ultimately delivers antigenic material to the class II MHC pathway. We review the role of Toll-like receptor 9 (TLR9) in phagosome maturation of Aspergillus fumigates–containing phagosomes. Advanced imaging modalities and the development of fungal like particles are promising tools that will aid in the dissection of the molecular mechanism to fungal immunity.
innate immunity; Dectin-1; Toll-like receptor; phagosome; macrophage; TLR9
Forkhead box P3 (FOXP3)+ is a transcription factor necessary for the function of regulatory T cells (Treg cells). Treg cells maintain immune homeostasis and self-tolerance, and play an important role in the prevention of autoimmune disease. Here, we discuss the role of Treg cells in the pathogenesis of myasthenia gravis (MG) and review evidence indicating that a significant defect in Treg cell in vitro suppressive function exists in MG patients, without an alteration in circulating frequency. This functional defect is associated with a reduced expression of key functional molecules such as FOXP3 on isolated Treg cells and appears to be more pronounced in immunosuppression-naive MG patients. In vitro administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the suppressive function of Treg cells and up-regulated FOXP3 expression. These findings indicate a clinically relevant Treg cell–intrinsic defect in immune regulation in MG that may reveal a novel therapeutic target.
myasthenia gravis; regulatory T cells; FOXP3; GM-CSF
The deep sequencing of an mRNA population, RNA-seq, is a very successful application of next-generation sequencing technologies (NGSTs). RNA-seq takes advantage of two key NGST features: (1) samples can be mixtures of different DNA pieces, and (2) sequencing provides both qualitative and quantitative information about each DNA piece analyzed. We recently used RNA-seq to study the transcriptome of Aspergillus fumigatus, a deadly human fungal pathogen. Analysis of the RNA-seq data indicates that there are likely tens of unannotated and hundreds of novel genes in the A. fumigates transcriptome, mostly encoding for small proteins. Inspection of transcriptome-wide variation between two isolates reveals thousands of single nucleotide polymorphisms. Finally, comparison of the transcriptome profiles of one isolate in two different growth conditions identified thousands of differentially-expressed genes. These results demonstrate the utility and potential of RNA-seq for functional genomics studies in A. fumigatus and other fungal human pathogens.
novel genes; annotation; population structure; differential expression; transcriptome profiling
Complement plays an important role in the pathophysiology of experimental autoimmune myasthenia gravis (EAMG). The deposition of IgG at the neuromuscular junction, followed by the activation and observance of C3 at the site, and finally the insertion of the membrane attack complex, which results in the destruction of the plasma membrane at the neuromuscular junction. Animal models’ of complement-deficient components show the importance of the mediated lysisin EAMG. These events have regulators that allow for the limitation in the cascade and the ability of the cell to inhibit complement at many places along the pathway. The complement regulatory proteins have many roles in reducing the activation of the complement cascade and the inflammatory pathways. Mice deficient in complement regulatory proteins, decay accelerating factor and CD59, demonstrate a significant increase in the destruction at the neuromuscular junction. Inhibition of complement-mediated lysis is an attractive therapeutic in MG.
complement; complement regulators; myasthenia gravis; C5; autoimmunity
Presynaptic active zones are synaptic vesicle release sites that playessential roles in the function and pathology of mammalian neuromuscular junctions (NMJs). The molecular mechanisms of active zone organization utilize presynaptic voltage-dependent calcium channels (VDCCs) in NMJs as scaffolding proteins. VDCCs interact extracellularly with the muscle-derived synapse organizer, laminin β2, and interact intracellularly with active zone-specific proteins, such as Bassoon, CAST/Erc2/ELKS2alpha, ELKS, Piccolo, and RIMs. These molecular mechanisms are supported by studies in P/Q- and N-type VDCCs double-knockout mice, and they are consistent with the pathological conditions of Lambert-Eaton myasthenic syndrome and Pierson syndrome, which are caused by autoantibodies against VDCCs or by a laminin β2 mutation. During normal postnatal maturation, NMJs maintain the density of active zones, while NMJs triple their size. However, active zones become impaired during aging. Propitiously, muscle exercise ameliorates the active zone impairment in aged NMJs, which suggests the potential for therapeutic strategies.
Bassoon; calcium channel; exercise; laminin; motor neuron; synapse
In myasthenia gravis (MG) and experimental autoimmune MG (EAMG), many pathologically significant autoantibodies are directed to the main immunogenic region (MIR) of muscle nicotinic acetylcholine receptors (AChRs), a conformation-dependent region at the extracellular tip of α1 subunits of AChRs. Human muscle AChR α1 MIR sequences were integrated into Aplesia ACh-binding protein (AChBP). The chimera potently induced EAMG. AChBP induced EAMG much less potently. AChBP is a water-soluble protein resembling the extracellular domain of AChRs, yet rats immunized with chimeras developed autoantibodies to both extracellular and cytoplasmic domains of muscle AChRs. We propose that an initial autoimmune response directed at the MIR leads to an autoimmune response sustained by muscle AChRs. Autoimmune stimulation sustained by endogenous muscle AChR may be a target for specific immunosuppression. These studies show that the α1 MIR is highly myasthenogenic, and that AChR-like proteins distantly related to muscle AChR can induce EAMG and, potentially, MG.
nicotinic acetylcholine receptor; AChR; MG; EAMG; antigenic structure
An appropriate density of acetylcholine receptors (AChRs) and Na+ channels (NaChs) in the normal neuromuscular junction (NMJ) determines the magnitude of safety factor (SF) that guarantees fidelity of neuromuscular transmission. In myasthenia gravis (MG), an overall simplification of the postsynaptic folding secondary to NMJ destruction results in AChRs and NaChs depletion. Loss of AChRs and NaChs accounts respectively for 59% and 40% reduction of the SF at the endplate, which manifests as neuromuscular transmission failure. The extraocular muscles (EOM) have physiologically less developed postsynaptic folding, hence a lower baseline SF, which predisposes them to dysfunction in MG and development of fatigue during “high performance” eye movements, such as saccades. However, saccades in MG show stereotyped, conjugate initial components, similar to normal, which might reflect preserved neuromuscular transmission fidelity at the NMJ of the fast, pale global fibers, which have better developed postsynaptic folding than other extraocular fibers.
safety factor; extraocular muscles; saccades; neuromuscular junction
During the past 5 years an increasing number of patients were diagnosed with congenital myasthenic syndromes (CMS) and a number of novel syndromes were recognized and investigated. This presentation focuses on the CMS caused by defects in choline acetyltransferase, novel fast-channel syndromes that hinder isomerization of the acetylcholine receptor from the closed to the open state, the consequences of deleterious mutations in the intermediate filament linker plectin, altered neuromuscular transmission in a centronuclear myopathy, and two recently identified CMS caused by congenital defects in glycosylation.
congenital myasthenic syndromes; acetylcholine receptor; fast-channel syndromes; choline acetyltransferase; plectin; centronuclear myopathy; GFPT1; DPAGT1
Biomarkers are defined as characteristics (proteins, RNA, single nucleotide polymorphisms, imaging) that are objectively measured and evaluated as an indicator of pathogenic processes or pharmacologic responses to a therapeutic intervention. Biomarkers are important in clinical trials where the robust biomarker reflects the underlying disease process in a sensitive and reliable manner. For myasthenia gravis (MG), acetylcholine receptor and muscle specific kinase antibodies, as well as single fiber electromyography, serve as excellent biomarkers for diagnosis but do not adequately substitute for clinical evaluations to predict treatment response. New technologies are emerging that enable broad biomarker discovery in biological fluids. Biomarker evaluation is ideally done in the context of longitudinal clinical trials. The MGTX trial has collected plasma and serum for RNA and protein analysis and thymus, which will allow robust biomarker discovery. The ultimate goal will be to identify candidates for a reliable substitute for a clinically meaningful endpoint that is a direct measure of the effectiveness of a therapy in the context of a continuum of disease natural history and a patient's overall well-being.
biomarkers; myasthenia gravis; surrogate endpoint; Prentice criteria
Prednisone is a frequently used treatment for myasthenia gravis (MG) but it has numerous side effects. Methotrexate is a selective inhibitor of dihydrofolate reductase and lymphocyte proliferation and is an effective immuosuppressive medication for autoimmune diseases. Given the negative results of the mycophenolate mofetil study, search for an effective immunosuppressant drug therapy is ongoing. The objective is to determine if oral methotrexate is safe and effective for MG patients who take prednisone. We have initiated a randomized, double-blind, placebo-controlled multicenter trial of methotrexate versus placebo in patients taking at least 10 mg/day of prednisone at enrollment. The methotrexate dose is increased to 20 mg and the prednisone dose is adjusted per protocol during the study. Clinical and laboratory evaluations are performed monthly for 12 months, with the primary efficacy measure being the nine-month prednisone area under the curve (AUC) from months 3 to 12. Secondary outcome measures include MG outcomes, quality of life measures, and a polyglutamation biomarker assay. A total of 18 U.S. sites and 2 Canadian sites are participating, with 48 screened cases, 42 enrolled, with 19 still active in the study.
methotrexate; myasthenia gravis; area under the curve; prednisone
The G4 motif, G≥3NxG≥3NxG≥3NxG≥3, is enriched in some genomic regions and depleted in others. This motif confers the ability to form an unusual four-stranded DNA structure, G4 DNA. G4 DNA is associated with genomic instability, which may explain depletion of G4 motifs from some genes and genomic regions. Conversely, G4 motifs are enriched downstream of transcription start sites, where they correlate with pausing. The uneven distribution of G4 motifs in the genome strongly suggests that mechanisms of selection act not only on one-dimensional genomic sequence, but also on structures formed by genomic DNA. The biological roles of G4 structures illustrate that, to understand genome function, it is important to consider the dynamic structural potential implicit in the G4 motif.
DNA; G-quadruplex; repeat; replication; transcription
In a medical sense biomodulation could be considered a biochemical or cellular response to a disease or therapeutic stimulus. In cancer pathophysiology, the initial oncogenic stimulus leads to cellular and biochemical changes that allow cells, tissue, and organism to accommodate and accept the oncogenic insult. In epithelial cell cancer development, the process of carcinogenesis is frequently characterized by sequential cellular and biochemical adaptations as cells transition through hyperplasia, dysplasia, atypical dysplasia, carcinoma in situ, and invasive cancer. In some cases, the adaptations may persist after the initial oncogenic stimulus is gone in a type of “hit-and-run” oncogenesis. These pathophysiological changes may interfere with cancer prevention therapies targeted solely to the initial oncogenic insult, perhaps contributing to resistance development. Characterization of these accommodating adaptations could provide insight for development of cancer preventive regimens that might more effectively biomodulate preneoplastic cells towards a more normal state.
cancer prevention; biomodulation; hit and run oncogenesis; viral oncogenesis; hormonal oncogenesis
Neurorestorative therapy targets multiple types of parenchymal cells in the intact tissue of the injured brain tissue to increase neurogenesis, angiogenesis, oligodendrogenesis, and axonal remodeling during recovery from neurological injury. In our laboratory, we tested thymosin β4 (Tβ4) as a neurorestorative agent to treat models of neurological injury. This review discusses our results demonstrating that Tβ4 improves neurological functional outcome in a rat model of embolic stroke, a mouse model of multiple sclerosis, and a rat model of traumatic brain injury. Tβ4 is a pleiotropic peptide exhibiting many actions in several different types of tissues. One mechanism associated with improvement of neurological improvement from Tβ4 treatment is oligodendrogenesis involving the differentiation of oligodendrocyte progenitor cells to mature myelin-secreting oligodendrocytes. Moreover, our preclinical data provide a basis for movement of Tβ4 into clinical trials for treatment of these devastating neurological diseases and injuries.
thymosin β4; stroke; multiple sclerosis; traumatic brain injury; rat
Obesity, a growing health problem worldwide, has been associated with the metabolic syndrome, diabetes, cardiovascular disease, hypertension, and other chronic diseases. Recently, the obesity–cancer link has received much attention. Epidemiological studies have shown that obesity is also associated with increased risk of several cancer types, including colon, breast, endometrium, liver, kidney, esophagus, gastric, pancreatic, gallbladder, and leukemia, and can also lead to poorer treatment and increased cancer-related mortality. Biological mechanisms underlying the relationship between obesity and cancer are not well understood. They include modulation of energy balance and calorie restriction, growth factors, multiple signaling pathways and inflammatory processes. Key among the signaling pathways linking obesity and cancer is the PI3K/Akt/mTOR cascade, which is a target of many of the obesity-associated factors and regulates cell proliferation and survival. Understanding the molecular and cellular mechanisms of the obesity–cancer connection is important in developing potential therapeutics. The link between obesity with cancer underscores the recommendation to maintain a healthy body weight throughout life as one of the most important ways to protect against cancer.
obesity; cancer; mechanisms; recommendations; prevention
Traumatic brain injury (TBI) remains a leading cause of mortality and morbidity worldwide. No effective pharmacological treatments are available for TBI because all Phase II/III TBI clinical trials have failed. This highlights a compelling need to develop effective treatments for TBI. Endogenous neurorestoration occurs in the brain after TBI, including angiogenesis, neurogenesis, synaptogenesis, oligodendrogenesis and axonal remodeling, which may be associated with spontaneous functional recovery after TBI. However, the endogenous neurorestoration following TBI is limited. Treatments amplifying these neurorestorative processes may promote functional recovery after TBI. Thymosin beta4 (Tβ4) is the major G-actin-sequestering molecule in eukaryotic cells. In addition, Tβ4 has other properties including anti-apoptosis and anti-inflammation, promotion of angiogenesis, wound healing, stem/progenitor cell differentiation, and cell migration and survival, which provide the scientific foundation for the corneal, dermal, and cardiac wound repair multicenter clinical trials. Here, we describe Tβ4 as a neuroprotective and neurorestorative candidate for treatment of TBI.
thymosin beta4; traumatic brain injury; rat; neuroprotection; neurorestoration
Heart failure (HF) is a major cause of mortality and morbidity, representing a leading cause of death and hospitalization among U.S. Medicare beneficiaries. Advances in science have generated effective interventions to reduce adverse outcomes in HF, particularly in patients with reduced left ventricular ejection fraction. Unfortunately, effective therapies for heart failure are often not utilized in an effective, safe, timely, equitable, patient-centered, and efficient manner. Further, the risk of adverse outcomes for HF remains high. The last decades have witnessed the growth of efforts to measure and improve the care and outcomes of patients with HF. This paper will review the evolution of quality measurement for HF, including a brief history of quality measurement in medicine; the measures that have been employed to characterize quality in heart failure; how the measures are obtained; how measures are employed; and present and future challenges surrounding quality measurement in heart failure.
quality measurement; heart failure; outcomes
Climate change is expected to lead to latitudinal and altitudinal temperature increases. High elevation regions such as the highlands of Africa, and those that have temperate climate are most likely to be affected. The highlands of Africa generally exhibit low ambient temperatures. This restricts the distribution of Anopheles mosquitoes, the vectors of malaria, filariasis and O’nyong’nyong fever. The development and survival of larval and adult mosquitoes are temperature dependent, as are mosquito biting frequency and pathogen development rate. Given that various Anopheles species are adapted to different climatic conditions, changes in the climate could lead to changes in species composition in an area which may change the dynamics of mosquito-borne disease transmission. It is important to consider the effect of climate change on rainfall which is critical to the formation and persistence of mosquito breeding sites. In addition, environmental changes such as deforestation could increase local temperatures in the highlands; this could enhance the vectorial capacity of the Anopheles. This experimental data will be invaluable in facilitating the understanding of the impact of climate change on Anopheles.
Hemorrhagic transformation (HT) associated with recombinant tissue plasminogen activator (rt-PA) complicates and limits its use in stroke. Here, we provide a focused review on the involvement of matrix metalloproteinase 9 (MMP-9) in rt-PA–associated HT in cerebral ischemia, and we review emerging evidence that the selective inhibitor of the sulfonylurea receptor 1 (Sur1), glibenclamide (U.S. adopted name, glyburide), may provide protection against rt-PA–associated HT in cerebral ischemia. Glyburide inhibits activation of MMP-9, ameliorates edema formation, swelling, and symptomatic hemorrhagic transformation, and improves preclinical outcomes in several clinically relevant models of stroke, both without and with rt-PA treatment. A retrospective clinical study comparing outcomes in diabetic patients with stroke treated with rt-PA showed that those who were previously on and were maintained on a sulfonylurea fared significantly better than those whose diabetes was managed without sulfonylureas. Inhibition of Sur1 with injectable glyburide holds promise for ameliorating rt-PA–associated HT in stroke.
rt-PA; Sur1; glyburide; MMP-9; cerebral ischemia; stroke
Certain chromosomal regions called common fragile sites are prone to difficulty during replication. Many tumors have been shown to contain alterations at fragile sites. Several models have been proposed to explain why these sites are unstable. Here we describe work to investigate models of fragile site instability using a yeast artificial chromosome carrying human DNA from a common fragile site region. In addition, we describe a yeast system to investigate whether repair of breaks at a naturally-occurring fragile site in yeast, FS2, involves mitotic recombination between homologous chromosomes, leading to loss of heterozygosity (LOH). Our initial evidence is that repair of yeast fragile site breaks does lead to LOH, suggesting that human fragile site breaks may similarly contribute to LOH in cancer. This work is focused on gaining understanding that may enable us to predict and prevent the situations and environments that promote genetic changes that contribute to tumor progression.
fragile site; FRA3B; flexibility peak; mitotic crossover; loss of heterozygosity; cancer
Gross chromosomal rearrangements (GCRs), or changes in chromosome structure, play central roles in evolution and are central to cancer formation and progression. GCRs underlie copy number variation (CNV), and therefore genomic disorders that stem from CNV. We study amplification in Escherichia coli as a model system to understand mechanisms and circumstances of GCR formation. Here, we summarize observations that led us to postulate that GCR occurs by a replicative mechanism as part of activated stress responses. We report that we do not find RecA to be downregulated by stress on a population basis and that constitutive expression of RecA does not inhibit amplification as would be expected if downregulation of RecA made cells permissive for nonhomologous recombination. Strains deleted for the genes for three proteins that inhibit RecA activity, psiB, dinI, and recX, all show unaltered amplification, suggesting that if they do downregulate RecA indirectly, it does not promote amplification.
nonhomologous recombination; amplification; stress; copy number variation