Raul Weiss, MD, FACC

• Director of the Electrophysiology Fellowship Program
• Richard M. Ross Heart Hospital
• Professor, Internal Medicine
• The Ohio State University Medical Center

As mentioned earlier pain and treatment center greensburg pa discount 100 mg cafergot fast delivery, a distinct form of amyloid is found in the brains of patients with Alzheimer disease pain treatment pregnancy order 100 mg cafergot otc. Amyloidosis of peripheral and autonomic nerves is a feature of several familial amyloidotic neuropathies neck pain treatment physiotherapy order cafergot with a visa. Depositions of amyloid in patients on long-term hemodialysis are most prominent in the carpal ligament of the wrist pain solutions treatment center reviews cafergot 100mg mastercard, resulting in compression of the median nerve (carpal tunnel syndrome) pain treatment center albany ky cheap cafergot 100mg with amex. Clinical Features Amyloidosis may be found as an unsuspected anatomic change hip pain treatment exercises purchase 100mg cafergot amex, having produced no clinical manifestations, or it may cause serious clinical problems and even death. The symptoms depend on the magnitude of the deposits and on the sites or organs affected. Clinical manifestations at first are often nonspecific, such as weakness, weight loss, lightheadedness, or syncope. Somewhat more specific findings appear later and most often relate to renal, cardiac, and gastrointestinal involvement. Itisseeninassociation with a variety of primary disorders, including monoclonal B-cell proliferations (in which the amyloid deposits consist of immunoglobulin light chains); chronic inflammatory diseases such as rheumatoid arthritis (deposits of amyloid A protein, derived from an acute-phase protein produced in inflammation); Alzheimer disease (amyloid protein); familial conditions in which the amyloid deposits consist of mutants of normal proteins. Pandey S, Kawai T, Akira S: Microbial sensing by Toll-like receptors and intracellular nucleic acid sensors. Merlini G, Dispenzieri A, Sanchorawala V et al: Systemic immunoglobulin light chain amyloidosis, Nat Rev Dis Primers 4:38, 2018. Even more agonizing than the mortality rate is the emotional and physical suffering inflicted by cancers. Some cancers, such as Hodgkin lymphoma, are curable, whereas others, such as pancreatic adenocarcinoma, are virtually always fatal. The only hope for controlling cancer lies in learning more about its causes and pathogenesis. Fortunately, great strides have been made in understanding its molecular basis, and some good news has emerged: cancer mortality for both men and women in the United States declined during the last decade of the 20th century and has continued its downward course in the 21st century. In this article, we describe the vocabulary of tumor biology and pathology and then review the morphologic characteristics that define neoplasia and allow benign and malignant tumors to be identified and distinguished. Also reviewed is the epidemiology of cancer, which provides a measure of the impact of cancer on human populations as well as clues to its environmental causes, insights that have led to effective prevention campaigns against certain cancers. Building on this foundation, we then discuss the biologic properties of tumors and the molecular basis of carcinogenesis, emphasizing the critical role that genetic alterations play in the development of neoplasia. Finally, we turn to cancer diagnosis, focusing on new technologies that are helping to direct the use of cancer drugs that are targeted at particular molecular lesions. Throughout, we give examples of new analytic methods and therapies that are not only changing our approach to cancer treatment but also providing new insights into cancer pathophysiology. Naming of benign tumors of mesenchymal cells is relatively simple; in general, the suffix "-oma" is attached to the name of the cell type from which the tumor arises. Thus a benign tumor of fibroblast-like cells is called a fibroma, a benign cartilaginous tumor is a chondroma, and so on. The nomenclature of benign epithelial tumors is more complex; some are classified based on their cell of origin, others on their microscopic appearance, and still others on their macroscopic architecture. Adenoma is applied to benign epithelial neoplasms derived from glandular tissues even if the tumor cells fail to form glandular structures. Thus, a benign epithelial neoplasm that arises from renal tubular cells and forms tightly clustered glands and a mass of adrenal cortical cells growing as a solid sheet are both referred to as adenomas. Benign epithelial neoplasms producing fingerlike or warty projections from epithelial surfaces are called papillomas, whereas those that form large cystic masses, such as in the ovary, are referred to as cystadenomas. Some tumors produce papillary projections that protrude into cystic spaces and are called papillary cystadenomas. When a neoplasm-benign or malignant- produces a grossly visible projection above a mucosal surface, for example, into the gastric or colonic lumen, it is termed a polyp. Tumor originally described swelling caused by inflammation, but is now equated with neoplasm. Although physicians know what they mean when they use the term neoplasm, it has been difficult to develop a precise definition. In the modern era, a neoplasm is defined as a genetic disorder of cell growth that is triggered by acquired or less commonly inherited mutations affecting a single cell and its clonal progeny. As discussed later, these causative mutations alter the function of particular genes and give the neoplastic cells a survival and growth advantage, resulting in excessive proliferation that is independent of physiologic growth signals and controls. All tumors are composed of two components: (1) neoplastic cells that constitute the tumor parenchyma and (2) reactive stroma made up of connective tissue, blood vessels, and cells of the adaptive and innate immune system. The classification of tumors and their biologic behavior are based primarily on the parenchymal component, but their growth and spread are critically dependent on their stroma. In others, parenchymal cells stimulate the formation of abundant collagenous stroma, referred to as desmoplasia. Some desmoplastic tumors-for example, some cancers of the female breast-are stony hard or scirrhous. Malignant Tumors Malignant tumors can invade and destroy adjacent structures and spread to distant sites (metastasize). Malignant tumors are collectively referred to as cancers, derived from the Latin word for crab, because they tend to adhere to any part that they seize on in an obstinate manner. Not all cancers pursue a deadly course; some are discovered at early stages Benign Tumors Benign tumors remain localized at their site of origin and are generally amenable to surgical removal. Nomenclature that allow for surgical excision, and others are cured with systemically administered drugs or therapeutic antibodies. The nomenclature of malignant tumors follows essentially the same schema used for benign neoplasms, with certain additions. Malignant tumors arising in solid mesenchymal tissues are usually called sarcomas (Greek sar = fleshy;. In squamous cell carcinoma the tumor cells resemble stratified squamous epithelium, whereas in adenocarcinoma the neoplastic epithelial cells grow in a glandular pattern. Sometimes the tissue or organ of origin can be identified and is added as a descriptor, as in renal cell adenocarcinoma or bronchogenic squamous cell carcinoma. In approximately 2% of cases, cancers are composed of cells of unknown origin and must be designated merely as undifferentiated malignant tumors. Small nests of epithelial cells and myxoid stroma forming cartilage and bone (an unusual feature) are present in this field. All of these elements arise from a single neoplastic clone capable of producing both epithelial and mesenchymal cells; thus the preferred designation of this neoplasm is pleomorphic adenoma. The great majority of neoplasms, including mixed tumors, are composed of cells from a single germ layer (mesoderm, endoderm, or ectoderm). An exception is a tumor called a teratoma, which contains recognizable mature or immature cells or tissues belonging to more than one germ cell layer (and sometimes all three). Teratoma originates from totipotential germ cells that are normally present in the ovary and testis and sometimes also found in abnormal midline embryonic rests. Such cells can differentiate into any cell type found in the body and so, not surprisingly, may give rise to neoplasms that contain, in a helter-skelter fashion, bone, epithelium, muscle, fat, nerve, and other tissues. For instance, the benign-sounding designations lymphoma, melanoma, mesothelioma, and seminoma are used for malignant neoplasms. Hamartomas are disorganized masses composed of cells indigenous to the involved tissue. For example, a small nodule of well-developed, normally organized pancreatic tissue may be found in the submucosa of the stomach or small intestine. The term choristoma, suggesting a neoplasm, imparts a gravity to these lesions that far exceeds their actual significance. Malignant tumors also tend to grow more rapidly than benign tumors, but there are so many exceptions that growth rate is not a reliable discriminator between benignity and malignancy. In fact, even cancers exhibit remarkably varied growth rates, from slow-growing tumors associated with survival for many years, often without treatment, to rapidly growing tumors that may be lethal within months or weeks. Blood Cells and Related Cell Types Muscle Smooth Striated Stratified squamous Basal cells of skin or adnexa Melanocytes Epithelial lining of glands or ducts Respiratory passages Renal epithelium Liver cells Urinary tract epithelium (transitional epithelium) Placenta epithelium Testicular epithelium (germ cells) Tumors of Epithelial Origin Differentiation and Anaplasia Differentiation refers to the extent to which neoplastic parenchymal cells resemble the corresponding normal parenchymal cells, both morphologically and functionally; lack of differentiation is called anaplasia. The neoplastic cells of a lipoma, a proliferation of benign adipocytes, may so closely resemble normal adipocytes as to be unrecognizable as a tumor by microscopic examination. This benign, well-differentiated tumor contains interlacing bundles of neoplastic smooth muscle cells that are virtually identical in appearance to normal smooth muscle cells in the myometrium. The tumor cells are strikingly similar to normal squamous epithelial cells, with intercellular bridges and nests of keratin pearls (arrow). In well-differentiated benign tumors, mitoses are usually rare and are of normal configuration. By contrast, most malignant neoplasms exhibit morphologic alterations that betray their potential for aggressive behavior. The malignant nature of such tumors is revealed by invasion of adjacent tissues and their ability to metastasize. Thus, cells within the same tumor are not uniform, but range from small cells with an undifferentiated appearance to tumor giant cells many times larger than their neighbors. These giant cells are not to be confused with inflammatory Langhans or foreign body giant cells, which are derived from macrophages and contain many small, normal-appearing nuclei. Characteristically, cancer cells have nuclei that are disproportionately large, with a nuclear-to-cytoplasm ratio that may approach 1: 1 instead of the normal 1: 4 to 1: 6. Note that compared with the well-formed and normal-looking glands characteristic of a benign tumor, the cancerous glands are irregular in shape and size and do not resemble the normal colonic glands. This tumor is considered moderately well differentiated because gland formation is seen. Metaplasia, Dysplasia, and Carcinoma In Situ these terms describe morphologically recognizable changes in differentiation that variously represent an adaptation to chronic injury (metaplasia), a premalignant change (dysplasia), or a cancer that has yet to invade (carcinoma in situ). Metaplasia is nearly always found in association with tissue damage, repair, and regeneration. Often the replacing cell type is better suited to some alteration in the local environment. For example, in Barrett esophagus, gastroesophageal reflux damages the squamous epithelium of the esophagus, leading to its replacement by glandular (gastric or intestinal) epithelium better suited to an acidic environment. The same is true of squamous metaplasia of the bronchial epithelium in chronic smokers, often a prelude to the development of lung cancer. Dysplastic cells may exhibit considerable pleomorphism and often contain large hyperchromatic nuclei with a high nuclear-to-cytoplasmic ratio. Dysplastic epithelial surfaces also typically show architectural disarray and a loss of orderly differentiation. For example, in dysplastic squamous epithelium the normal progressive maturation of tall cells in the basal layer to flattened squames on the surface may fail in part or entirely, leading to replacement of the epithelium by basal-like cells with hyperchromatic nuclei. In addition, mitotic figures are more abundant than in the normal squamous epithelium and may be seen throughout dysplastic epithelium, rather than being confined to the basal layer, as is the normal case. In situ epithelial cancers display all of the cytologic features of malignancy and unless treated have high probability of progression to invasive cancers. Dysplastic changes are often found adjacent to foci of invasive carcinoma, and in some situations, such as in the cervix, severe epithelial dysplasia or carcinoma in situ frequently antedates the appearance of cancer. Moreover, some mutations associated with full-blown cancer (described later) may be present in even "mild" dysplasias. Nevertheless, although dysplasia may be a precursor to malignant transformation, it does not always progress to cancer. With removal of inciting causes, even moderately severe dysplasias may be completely reversible. Note the marked cellular and nuclear pleomorphism, hyperchromatic nuclei, and tumor giant cells. Unlike benign tumors and some well-differentiated malignant neoplasms, undifferentiated cancers often contain many cells in mitosis, reflecting their high rate of proliferation. For example, cells in mitosis are often seen in normal tissues exhibiting rapid turnover, such as the epithelial lining of the gut and nonneoplastic proliferations such as hyperplasias. In addition to cytologic abnormalities, the orientation of anaplastic cells with respect to each other or to supporting structures like basement membranes is markedly disturbed. While growing tumor cells must have a blood supply, the vascular stroma is often insufficient; as a result, many rapidly growing cancers develop areas of ischemic necrosis. As one might surmise, well-differentiated transformed cells have a greater likelihood of retaining the functional capabilities of their normal counterparts. Benign tumors are almost always well differentiated and often retain normal functions, as do many well-differentiated cancers.

Absence of cell-mediated immunity is caused by low numbers of T lymphocytes in the blood and lymphoid tissues and poor defense against certain fungal and viral infections pain treatment centers of america colorado springs buy cafergot from india. The T-cell zones of lymphoid organs-paracortical areas of the lymph nodes and the periarteriolar sheaths of the spleen-are depleted pain medication for dogs tylenol purchase cafergot 100mg with mastercard. Ig levels may be normal or reduced cape fear pain treatment center pa buy cafergot 100 mg without a prescription, depending on the severity of the T-cell deficiency pain treatment lexington ky best buy cafergot. In the vast majority (90%) of cases pain management dogs cats purchase cafergot overnight delivery, DiGeorge syndrome is caused by a small germline deletion that maps to chromosome 22q11 pain treatment and research generic cafergot 100 mg visa, and DiGeorge syndrome is now considered a component of the 22q11 deletion syndrome, discussed in Chapter 5. As an X-linked disease, this disorder is seen almost exclusively in males, but sporadic cases have been described in females, possibly caused by mutations in other genes that function in the same pathway. The disease usually does not become apparent until about 6 months of age, as maternal immunoglobulins are depleted. In most cases, recurrent bacterial infections of the respiratory tract, such as acute and chronic pharyngitis, sinusitis, otitis media, bronchitis, and pneumonia, call attention to the underlying immune defect. Almost always the causative organisms are Haemophilus influenzae, Streptococcus pneumoniae, or Staphylococcus aureus. Because antibodies are important for neutralizing infectious viruses, individuals with this disease are also susceptible to certain viral infections, especially those caused by enteroviruses, such as echovirus, poliovirus, and coxsackievirus. These viruses infect the gastrointestinal tract, and from here they can disseminate to the nervous system via the blood. Thus, immunization with live poliovirus carries the risk of paralytic poliomyelitis, and echovirus can cause fatal encephalitis. For similar reasons, Giardia lamblia, an intestinal protozoan that is normally resisted by secreted IgA, causes persistent infections in persons with this disorder. Paradoxically, autoimmune diseases, such as arthritis and dermatomyositis, occur in as many as 30% of individuals with this disease. It is likely that these autoimmune disorders are caused by a breakdown of self-tolerance resulting in autoimmunity, but chronic infections associated with the immune deficiency may play a role in inducing the inflammatory reactions. The treatment of X-linked agammaglobulinemia is replacement therapy with immunoglobulins. Other Defects in Lymphocyte Maturation Many other rare causes of immunodeficiency resulting from defective lymphocyte maturation have been documented. Other defects are caused by mutations in antigen receptor chains or signaling molecules involved in T- or B-cell maturation. Hyper-IgM Syndrome In this disorder, affected patients have IgM antibodies but are deficient in IgG, IgA, and IgE antibodies. This interaction triggers Ig class switching and affinity maturation in B cells, and also stimulates the microbicidal functions of macrophages. In the remaining patients, the disease is inherited in an autosomal recessive pattern. The serum of persons with this syndrome contains normal or elevated levels of IgM but no IgA or IgE and extremely low levels of IgG. Clinically, patients present with recurrent pyogenic DiGeorge Syndrome (Thymic Hypoplasia) DiGeorge syndrome is a T-cell deficiency that results from failure of development of the thymus. The third and fourth pharyngeal pouches, which give rise to the thymus, the parathyroids, some of the C cells of the thyroid, and Immunodeficiency diseases infections, because the level of opsonizing IgG antibodies is low, and also because affinity maturation, a process necessary for production of high-affinity antibodies, is impaired. Occasionally, the IgM antibodies react with blood cells, giving rise to autoimmune hemolytic anemia, thrombocytopenia, and neutropenia. In older patients, there may be a proliferation of IgM-producing plasma cells that infiltrates the mucosa of the gastrointestinal tract. In the United States, it occurs in about 1 in 600 individuals of European descent. Because IgA is the major antibody in mucosal secretions, mucosal defenses are weakened, and infections occur in the respiratory, gastrointestinal, and urogenital tracts. Symptomatic patients commonly present with recurrent sinopulmonary infections and diarrhea. The basis of the increased frequency of autoimmune and allergic diseases is not known. When transfused with blood containing normal IgA, some patients develop severe, even fatal, anaphylactic reactions, because the IgA behaves like a foreign antigen. Common Variable Immunodeficiency this relatively frequent entity encompasses a heterogeneous group of disorders in which the common feature is hypogammaglobulinemia, generally affecting all the antibody classes but sometimes only IgG. The diagnosis of common variable immunodeficiency is based on exclusion of other well-defined causes of decreased antibody production. Relatives of such patients have a high incidence of selective IgA deficiency (see later), suggesting that at least in some cases, selective IgA deficiency and common variable immunodeficiency represent different expressions of a common genetic defect in antibody synthesis. In contrast to X-linked agammaglobulinemia, most individuals with common variable immunodeficiency have normal or near-normal numbers of B cells in the blood and lymphoid tissues. The clinical manifestations of common variable immunodeficiency are caused by antibody deficiency, and hence they resemble those of X-linked agammaglobulinemia. Individuals with this disorder are also prone to the development of persistent diarrhea caused by G. Common variable immunodeficiency affects both sexes equally, and the onset of symptoms is later than in X-linked agammaglobinemina, in childhood or adolescence. As in X-linked agammaglobulinemia, these patients have a high frequency of autoimmune diseases (approximately 20%), including rheumatoid arthritis. The risk of lymphoid malignancy is also increased, and an increase in gastric cancer has been reported. Other Defects in Lymphocyte Activation Many rare cases of lymphocyte activation defects have been described, affecting antigen receptor signaling and various biochemical pathways. Mutations affecting Th1 responses are associated with atypical mycobacterial infections; the syndrome is called Mendelian susceptibility to mycobacterial disease. Inherited defects in Th17 responses lead to chronic mucocutaneous candidiasis and bacterial infections of the skin (a disorder called Job syndrome). Immunodeficiencies Associated With Systemic Diseases In some inherited systemic disorders, immune deficiency is a prominent clinical problem. Wiskott-Aldrich Syndrome Wiskott-Aldrich syndrome is an X-linked disease characterized by thrombocytopenia, eczema, and a marked vulnerability to recurrent infection, resulting in early death. The thymus is morphologically normal, at least early in the course of the disease, but there is progressive loss of T lymphocytes in the peripheral blood and in the T-cell zones (paracortical areas) of the lymph nodes, with variable defects in cellular immunity. Patients do not make antibodies to polysaccharide antigens, and the response to protein antigens is poor. Ataxia Telangiectasia Ataxia telangiectasia is an autosomal-recessive disorder characterized by abnormal gait (ataxia), vascular malformations (telangiectases), neurologic deficits, increased incidence of tumors, and immunodeficiency. The immunologic defects are of variable severity and may affect both B and T cells. The most prominent humoral immune abnormalities are defective production of isotype-switched antibodies, mainly IgA and IgG2. The T-cell defects, which are usually less pronounced, are associated with thymic hypoplasia. Patients experience upper and lower respiratory tract bacterial infections, multiple autoimmune phenomena, and increasingly frequent cancers with advancing age. Like several other immunodeficiency syndromes, patients with ataxia telangiectasia have a markedly increased incidence of lymphoma. Secondary Immunodeficiencies Secondary (acquired) immune deficiencies may be encountered in individuals with cancer, diabetes and other metabolic diseases, malnutrition, chronic infection, and in persons receiving chemotherapy or radiation therapy for cancer, or immunosuppressive drugs to prevent graft rejection or to treat autoimmune diseases (Table 6. As a group, the secondary immune deficiencies are more common than the disorders of primary genetic origin. Some of these secondary immunodeficiency states can be caused by defective lymphocyte maturation (when the bone marrow is damaged by radiation or chemotherapy or involved by tumors, such as leukemias), inadequate Ig synthesis (as in malnutrition), or lymphocyte depletion (from drugs or severe infections). Because of public health measures, the infection rate seems to be decreasing, and some authorities believe it may have peaked in the late 1990s. Furthermore, improved antiviral therapies have resulted in fewer people dying of the disease. However, these newer treatments are not readily available in many low income countries, and toxic side effects remain a problem. Heterosexual spread of the virus is occurring most rapidly in female sex workers and in women in long-term marital or cohabitating relationships, particularly among adolescents. Intravenous drug users with no previous history of homosexuality are the next largest group, representing about 20% of infected individuals. The three major routes of transmission are sexual contact, parenteral inoculation, and passage of the virus from infected mothers to their newborns. Because the majority of infected people in the United States are men who have sex with men, most sexual transmission has occurred among homosexual men. In addition to male-to-male and male-to-female transmission, female-to-male transmission also occurs. Currently, this risk is estimated to be 1 in more than 2 million units of blood transfused. Infected mothers can transmit the infection to their offspring by three routes: (1) in utero by transplacental spread, (2) during delivery through an infected birth canal, and (3) after birth by ingestion of breast milk. Of these, transmission during birth (intrapartum) and in the immediate period thereafter (peripartum) is considered to be the most common mode in the United States. The reported transmission rates vary from 7% to 49% in different parts of the world. Seroconversion has been documented after accidental needle-stick injury or exposure of nonintact skin to infected blood in laboratory accidents. After needlestick accidents, the risk of seroconversion is believed to be about 0. The viral particle is covered by a lipid bilayer derived from the host cell and studded with viral glycoproteins gp41 and gp120. The viral core is surrounded by a matrix protein called p17, which lies underneath the virion envelope. The products of the gag and pol genes are large precursor proteins that are cleaved by the viral protease to yield the mature proteins. For example, the product of the tat (transactivator) gene causes a 1000-fold increase in the transcription of viral genes and is critical for virus replication. Included in this group are feline immunodeficiency virus, simian immunodeficiency virus, visna virus of sheep, bovine immunodeficiency virus, and the equine infectious anemia virus. The infection becomes established in lymphoid tissues, where the virus may remain latent for long periods. We first describe the mechanisms involved in viral entry into T cells and macrophages and the replicative cycle of the virus within cells. The molecules and mechanisms of each of these steps are understood in considerable detail. R5 strains preferentially infect cells of the monocyte/macrophage lineage and are thus referred to as M-tropic, whereas X4 strains are T-tropic, preferentially infecting T cells. Over the course of infection, however, T-tropic viruses gradually accumulate; these are especially virulent because T-tropic viruses are capable of infecting many T cells and even thymic T-cell precursors and cause greater T-cell depletion and impairment. Only rare homozygotes for the mutation have been found in African or East Asian populations. After integration, the provirus may be silent for months or years, a form of latent infection. Such productive infection, when associated with extensive viral budding, leads to death of infected cells. In addition to direct killing of cells by the virus, other mechanisms may contribute to the loss of T cells. During this process, inflammatory cytokines and cellular contents are released, thus potentiating recruitment of new cells and increasing the numbers of cells that can be infected. These include reduced antigen-induced T-cell proliferation, decreased Th1-type responses, defects in intracellular signaling, and many more. The loss of Th1 responses results in a profound deficiency of cellmediated immunity, leading to increased susceptibility to infections by viruses and other intracellular microbes. It is widely believed that integrated provirus, without viral gene expression (latent infection), can remain in the cells for months to years. Because the frequency of infected cells in the circulation is very low, for many years it was suspected that the immunodeficiency is out of proportion to the level of infection and cannot be attributed to death of infected cells. Therefore, loss of this "master regulator" has ripple effects on virtually every other component of the immune system, as summarized in Table 6. In certain tissues, such as the lungs and brain, as many as 10% to 50% of macrophages are infected. Paradoxically, early in the disease course there is polyclonal activation of B cells, resulting in germinal center B-cell hyperplasia, sometimes accompanied by autoimmune phenomena like immune thromoboctyopenic purpura. Plasma cells also increase in number, leading to hypergammaglobulinemia and bone marrow plasmacytosis. This could be due, in part, to lack of T-cell help, but antibody responses against T-independent antigens are also suppressed, and hence there may be intrinsic defects in B cells as well.

The application of genetic and molecular biological tools to the study of oral microbiology now includes important oral bacterial genera ankle pain treatment running purchase cafergot now, such as Aggregatibacter pain treatment doctors 100mg cafergot with visa, Porphyromonas midsouth pain treatment center jackson tn cheap cafergot 100 mg online, Actinomyces allied pain treatment center youngstown oh discount cafergot 100mg with amex, Prevotella joint pain treatment options generic cafergot 100mg otc, Fusobacterium knee pain treatment without surgery cafergot 100mg low cost, and Treponema. With the availability of the genetic and molecular tools described in this chapter, it is possible to confirm a function Genetics and Molecular Biology of Oral Microorganisms 187 predicted by the sequence of a gene, to examine environmental conditions that regulate its expression, or to elucidate the function in each species of the 30 to 40% of genes whose function cannot be predicted by sequence alone. Thus, transposons can be used for random mutagenesis where screening for a phenotypic property is possible. A bacterial mutant in which only a defined gene has been altered is termed an isogenic mutant; such alteration will lead to a change or loss of function specific to that gene. Restoration of the gene and/ or its expression, referred to as complementation of the mutant, should restore function. A single recombination (or crossover) event will result in integration of the entire plasmid and disruption of the gene. Markerless allelic replacement strategies have also been devised that use temperature-sensitive or counterselectable vectors. In all cases, it is important to assess any potential efects on genes downstream of the target gene. These vectors can be used to generate double recombination mutants or to provide an intact copy of a gene for complementation. Restoration of gene function by complementation ensures that the phenotypic efect observed was due to inactivation of the gene and not to a polar efect on a downstream gene or other unintended mutation. Regulation of competence in Gram-negative bacteria can occur by catabolite repression. Plasmids must encode the elements required for autonomous replication, including an origin of replication (ori) and contiguous gene(s) encoding replication proteins and other controlling elements. Other phenotypes that may be encoded by plasmids include virulence traits, metabolic capabilities, or the production of bacteriocins. Mobilizable plasmids require genes encoding conjugative functions to be provided by another plasmid or the chromosome. The impact of horizontal gene transfer on the adaptive ability of the human oral microbiome. The past decade has ushered in the "multi-omics" revolution, which has dramatically reshaped our understanding of host-microbe interactions and has provided new insights into the mechanisms by which bacteria interact with and colonize the host and cause disease. These advances have also led to the development of molecular diagnostic protocols for more rapid identifcation of oral microbial taxa and appropriate molecular therapeutic targets. The frst two complete genome sequences for the oral bacteria Strepto coccus mutans and Fusobacterium nucleatum were published in 2002. High-throughput methods are now frequently used for genome-wide analysis to study genotypephenotype relationships, for more precise ecological analyses, and for genetic characterization of the oral bacterial metagenome. It has recently manifested as a simple and effcient alternative to classical gene-editing techniques in bacteria. In this article, we describe some of the fundamental processes in bacterial gene regulation with particular emphasis on oral bacteria. The continued progress and success of these technologies will bring many pragmatic benefts, particularly in therapeutics for oral and related systemic infections. The genome of an organism consists of its chromosomes and any extrachromosomal elements that may be present. The completed genome sequences of a multitude of microorganisms are available for analysis through the internet. Given the ease with which genomes may now be sequenced, many thousands of bacterial genomes will be sequenced over the next few years. A bacterial species is defned by its pan-genome, which includes the full complement of genes in a particular species of bacteria. The core genome contains the set of genes required for basic cellular functions, and these are present in all strains of the same bacterial species. The majority of genes making up the core genome belong to groups with housekeeping functions, many of which are essential genes, for example, those encoding proteins involved in cell envelope/cell wall metabolism, nutrient utilization, and global gene regulation. Core genes are not subject to frequent horizontal transfer and generally do not contribute to the adaptive evolution of bacterial pathogens. Identifying the core complement of genes in a bacterial species is often the frst step in population genomics studies. The variable genome includes the genes that are differentially present between individual strains or unique to a given strain. Some genes of the variable genome may play a role in adaptation to special growth conditions, such as those involved in the colonization of new ecological niches, symbiosis, host cell interactions, and pathogenicity. In addition, many of the strain-specifc genes encode hypothetical or unknown function proteins. A total of 86% of W83 genes are part of the core genome, and 14% of the genes comprise the variable region. The plasticity of the variable genome contributes to bacterial genome evolution during which the genomic sequences undergo different changes. Many of the changes (such as singlenucleotide variation, insertion/deletion, and copy number change) arise spontaneously, and the observed genomic changes can either be distributed uniformly throughout the genome or be preferentially localized to some regions compared to others. Selection pressure, such as through drugs or immune action, plays an important role in determining genomic regions likely to harbor hot spots. Gene Organization and Regulation A bacterial genome usually contains thousands of genes, which are not all expressed at the same time. Most of these genes have specifc roles and are expressed only in certain metabolic states. The gene encoding the repressor protein may be located near the operon or elsewhere in the chromosome. Some operons are inducible in that they can only be turned on or expressed in the presence of a specifc molecule known as inducer. Others are repressible, meaning that their expression can be downregulated or turned off once the specifc repressor protein is present. Such tightly regulated gene expression systems ensure that proteins are expressed only when needed for relevant functions. The lac operon contains three structural genes that encode proteins required for lactose transport and metabolism, namely, lacZ, lacY, and lacA. The lacZ gene encodes -galactosidase, which converts lactose into glucose and galactose; the lacY gene encodes -galactoside permease, which transports lactose into the cell; and the lacA gene codes for -galactoside transacetylase. The transacetylase is not crucial for metabolism of lactose but plays a role in the detoxifcation of components that may come inside the cell during lactose transport. So together, these gene products frst transport lactose into the cell from the local environment and then break it down to monosaccharides for use as an energy source. These include a single promoter upstream of three lac genes, an operator sequence between the promoter and structural genes that helps determine if transcription will take place, and a terminator that is located downstream of lac structural genes and tells the transcriptional machinery to terminate transcription. One more important regulatory element is the lac regulatory gene (lacI), which is found just outside the lac operon and has its own promoter and terminator sequences. The lac operon also has a second control mechanism that is in response to glucose concentration. When both lactose and glucose are present, the bacteria will preferentially utilize glucose before the lac operon is turned Applied Molecular Biology and the Oral Microbes 193 on, because glucose is a monosaccharide and more easily metabolized. The trp operon contains a promoter, an operator located at the 3 end of the promoter, along with fve structural genes-trpE, trpD, trpC, trpB, and trpA-which are required for the biosynthesis of tryptophan. The repressor TrpR protein is encoded by the trpR regulator gene, which is not part of the trp operon and is located elsewhere in the bacterial chromosome. In fact, bacteria always have antitermination mechanisms that inhibit the functions of the terminator and termination factors resulting in increased expression of downstream genes. Why such a central process should be ineffcient is not clear, although several instances of transcriptional readthrough have been reported to beneft the cell. These are known as "orphan response regulators," as they lack a cognate histidine kinase as a phosphorylation partner. Transcriptional Initiation Most bacterial gene regulation takes place at the transcriptional level (see Table 1). The two main elements are the -10 and the -35 hexamers, which are located 10 and 35 bp upstream from the transcription start site (+1), respectively. In presence of lactose, the operon is induced by lactose metabolite allolactose to express lactose-metabolizing enzymes. Allolactose binds to the LacI repressor, making it inactive so that it loses the ability to bind to the lac operator. The -10 and -35 sequences are conserved on average but are not found intact in all promoters. On average, only three or four of the six base pairs in each consensus sequence are found in any given promoter. In fact, there are very few natural promoters that exactly match the consensus sequence. In general, the closer the -10 and -35 sequences of a promoter are to the consensus sequence, the stronger is the promoter. While the general features of promoter design are conserved across bacterial species, it is important to note that for Grampositive bacteria and spirochetes, the upstream regulatory sequences may not conform either to consensus sequences or positioning as described for E. In bacteria, gene transcription is often controlled by availability of metal ions. These are critical for many reactions (such as cofactors for the function of the enzymes involved in electron transport and oxidative stress responses), but excess metals can be toxic. Therefore, bacteria must have mechanisms to regulate intracellular metal ion levels and maintain homeostasis. As bacteria cannot synthesize or degrade metal ions, the balance of metal ions mainly depends upon their transport into and out of the cell. Whenever the level of a particular metal ion decreases below a certain limit, bacteria activate pathways involved in the import and mobilization of metals, whereas when there is an excess of metal ions, efflux and storage pathways are induced. Bacteria respond to metal ion limitation or excess by regulating the expression of specifc genes controlled by metal-sensing transcription factors, also known as metalloregulatory proteins. A typical example is Fur (ferric uptake regulator), a global regulator family protein controlling iron homeostasis in many bacteria, including P. In the presence of suffcient intracellular iron concentration, the dimeric Fur protein binds to two Fe2+ ions, which allows it (Fur-Fe2+) to bind a specifc 19-bp "Fur box" in the promoter region of iron-regulated genes, resulting in repression of genes that function in metal intake. Similar to Fur, Zn2+ ions are sensed by the zinc uptake regulator (Zur), and Mn2+ is sensed by the MntR regulator. SloR is a homodimeric, manganese-dependent transcriptional repressor protein in S. Streptococcal species coordinate the control of metal ion homeostasis with levels of oxidative stress, bioflm formation, and virulence properties. For example, genes that encode iron-scavenging proteins are usually upregulated in response to low environmental iron concentrations. However, the activities of many bacterial gene products are generally diffcult to assay or detect, unless they have easily measurable enzymic activity or a specifc antibody to the protein product is available. Thus, surrogate genes, referred to as "reporter genes," which encode well-studied and easily assayed enzymes, are often used in regulation studies. To assess the effects of specifc environmental signals (or any parameter) on the expression of a gene of interest, a construct is designed such that a reporter gene is incorporated immediately downstream of (and at times as a replacement for) the gene of interest. The result is a transcriptional fusion in which the regulatory region of the gene of interest now controls the expression of the reporter gene. This type of fusion is produced by cloning a promoterless reporter gene downstream of the promoter of the gene of interest. One of the more common reporter genes used to study gene regulation in oral microorganisms is lacZ (which encodes -galactosidase; see above). When a chromogenic substrate is provided in place of the normal substrate lactose, LacZ activity can be measured by a simple color-change assay. Another commonly used reporter gene is cat, which encodes chloramphenicol acetyltransferase, an enzyme that inactivates the antibiotic chloramphenicol via the addition of an acetyl group to the molecule. The reporter is particularly useful because the activity of the enzyme can be measured directly or by determining the level of antibiotic resistance. Other useful reporter genes include galK, a gene whose product galactokinase phosphorylates galactose. Galactokinase activity can be measured by a radioactive assay using galactose and radiolabeled phosphorus as substrates. The activities of fluorescent and chemiluminescent proteins can be detected visually and can also be quantitated with specialized instruments, such as fluorimeters. Although multiple approaches are available, their adaptation for use in oral microorganisms has been generally limited. The lack of availability of animal models is one limitation for the study of host-induced genes of oral pathogens. Additionally, in those cases for which an animal model is available, it is often not a close approximate to the human condition. The use of pooled sera allows the identifcation of the widest possible array of antigens produced during different stages of infection and, when appropriate, from patients infected via different routes. The mining of genomic sequences for genes that encode potential virulence factors and other functions of interest may be conducted by several approaches. Phyre2 (pronounced "fre") is a suite of tools available on the web to predict and analyze protein structure and function. Phyre2 uses advanced remote homology detection methods to build three-dimensional models and predict ligand-binding sites, among other functions. Structural prediction accuracy is based on protein structure being more evolutionarily conserved than protein sequence and the fnite and relatively small number of unique protein folds in nature. Like many similar algorithms, it utilizes pattern recognition systems such as hidden Markov models for its predictions. It is important to stress, however, that genes annotated on the basis of sequence similarities do not necessarily encode proteins with similar functions.

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