Sunday, December 2, 2018

Control of Inflammation following Injury or Infection

The natural immune responses that are elicited after an insult to the body as a result of injury or infection from a pathogen, is both rapid and powerful. Initially inflammatory cells are mobilized to the site of the trauma. The predominant cell types that are recruited are macrophages and neutrophils that release free radical reactive oxygen and nitrogen species (RONS) that are lethal to invading organisms. However, these chemical moieties are so powerful that they can also kill and mutate the surrounding normal tissue. Although there are naturally induced anti-inflammatory responses that are also initiated, the optimum balance is difficult to achieve. In some diseases, such as ulcerative colitis and rheumatoid arthritis, such anti-inflammatory responses are unavailable. Therefore, for this reason medical intervention in the form of medication becomes appropriate. 

There are, of course, many different anti-inflammatory medications currently available. Recently Dr. Torkild Visnes and his colleagues from the Karolinska Institutet in Sweden in collaboration with the University of Texas Medical Branch, Uppsala University and Stockholm University have discovered a new methodology for enhancing the anti-inflammatory response. In order to understand the investigator’s approach, we need to examine the rationale for this research in greater detail. 

Normally the enzyme 8-oxyguanine DNA glycosylase 1 (OGG1) functions as a DNA repair enzyme that both recognizes and repairs the nucleotide base excision repair of 7,8 dihydro-8-oxoguanine (8-oxoG) that represents one of the major types of DNA damage produced by RONS. Paradoxically, the binding of OGG1 to 8-oxoG, facilitates the action of the NF-kB transcription factor that promotes the activation of quiescent chemokine and cytokine genes that subsequently leads to the inflammatory response and the subsequent release of RONS at the site of trauma. 

Visnes and his group have identified a small molecule (TH5487) that binds to the active site of OGG1 and effectively blocks its repair capabilities on account of the fact that inhibited OGG1 cannot bind to that G-rich region of the DNA leads to the activation of the NF-kB transcription factor that promotes proinflammatory genes. In fact, TH5487 has been shown to inhibit this process in mouse and human lung epithelial cells in vitro and the TNF-induced neutrophil inflammation in the in vivo mouse model. 

This research is of value since it elucidates a molecular mechanism that demonstrates a connection between the normal DNA repair function of OGG1 and the inflammatory response and has discovered a small molecule inhibitor of this process.

Saturday, October 6, 2018

RNA Modifications Regulate Gene Expression During Development

RNA plays a critical role in gene expression within the cellular environment. Both messenger RNA (mRNA) and transfer RNA (tRNA) are involved in translating the instructions encoded in the DNA sequence of expressed genes into the manufacture of protein products at the site of the ribosomes. This process is, of course, happening continually throughout the life cycle of the cell. In the development of the human embryo in utero, the timing and orchestration of gene expression is vitally important; the precision of these processes is an absolute requirement for the successful creation of a viable individual at birth. The timed expression of development-related genes is determined by gene expression programs.

There is current evidence that there are modifications of RNA that function as post-transcriptional regulators of these gene expression programs. These regulators apparently impact a wide variety of eukaryotic biological processes. Michaela Frye from the Department of Genetics at the University of Cambridge, UK and her scientific collaborators stated in a recent article in the publication, Science, that “N6-methyladenosine affects the translation and stability of the modified transcripts, thus providing a mechanism to coordinate the regulation of groups of transcripts during cell state maintenance and transition. Similarly, some modifications in transfer RNAs are essential for RNA structure and function. Others are deposited in response to external cues and adapt global protein synthesis and gene-specific translation accordingly and thereby facilitate proper development.”


Gene expression in multicellular organisms is determined by a complex set of interacting and dynamic processes that require the coordination of mRNA metabolism and protein synthesis. There has been considerable investigation into the mechanism of transcriptional networks related to tissue-specific stem cell differentiation. However, the regulation of gene expression programs during development is especially crucial and must be unerringly precise. The recent evidence points to specific modifications of RNA as crucial to the regulation of cellular transcriptomes and proteomes during development.

At present there are over 170 modifications in RNA reported, but it is only following the relatively recent development of precise analytical tools that these modifications can be identified and quantified with precision. In addition to the N6 methyladenosine modification of mRNA as a crucial regulator of gene expression, other modifications including 5-methylcytosine and N1-methyladenosine are involved in the modification of both noncoding RNA and mRNA.

A detailed examination of some of these changes has been reported in the Science publication authored by Michaela Frye as noted above. These findings help to elucidate the molecular and cellular mechanisms that are involved in the intricate process of development.

Sunday, July 29, 2018

An Elegant and Monumental Experiment

The year is 1952 – two years prior to the discovery of the three-dimensional structure of DNA.  At that time there were essentially two schools of thought regarding the class of organic compounds responsible for heredity, namely, proteins or nucleic acids.  Two researchers Alfred Hershey (1908 – 1997) and his assistant Martha Chase (1923 – 2003) collaborated on a research project that they felt could unambiguously provide the answer to this fundamental question.  The experiment described below is remarkably simple, precise and elegant; the ramifications of their results speaks for itself in regards to the discoveries that would follow including Watson and Crick’s elucidation of the structure DNA and ultimately the complete sequence of the human genome (2003).

Hershey and Chase focused their attention upon the bacteriophage – a type of virus that preferentially attacks bacterial cells.  Like all viruses, the phage is made up of two distinct classes of compounds – a protein coat that surrounds a DNA core (there is also a class of viruses that use RNA as the infecting agent, the AIDS virus, for example).

Their experimental approach involved two precise steps outlined below
  •          They labeled the phage (T2) with radioactive Phosphorus (P32) prior to introducing T2 to bacterial host.  Phosphorus is predominantly found in DNA where it is a major constituent and found in protein in insignificant amounts.  After infection, they found that P32 was no longer in the phage, but was found in the host indicating the phage DNA was transferred to the host. 
  •          In the second experiment, they preferentially labeled the phage protein with radioactive Sulfur (S35).  Sulfur is a significant part of the composition of proteins but does not exist in nucleic acids (DNA).  In their analysis subsequent to infection, they found that S35 remained with the phage but was not found in the bacterium.

The results from these experiments, clearly demonstrated that the infectious agent was DNA and not protein.  This conclusion was so significant at the time that James Watson stated that, “the Hershey-Chase experiment had a much broader impact than most confirmatory announcements and made me ever more certain that finding the three-dimensional structure of DNA was biology's next important objective.

Note, that Hershey won the Nobel Prize for his work in 1969 along with Max Delbruck and Salvador Luria.  This kind of experimental approach also demonstrates the roles that imagination, dedication, persistence and creativity play in conducting scientific research. 

Saturday, July 7, 2018

A Rationale for the High Mortality Rate of Pancreatic Cancer

Pancreatic cancer is an especially aggressive cancer with a high mortality rate – only 6% of those affected survive beyond 5 years.  It is also the fourth most common death from cancer worldwide.  Even under conditions when it was supposedly caught early on, the adenocarcinoma was successfully resected and the liver was deemed to be free of the presence of metastatic legions, patients, nonetheless suffered from subsequent metastatic disease.  The obvious conclusion from this observation is that there are latent metastases that persist and that are only detectable microscopically.
Furthermore, these latent metastases were believed to represent a balance between cancer cell growth and cancer cell death precipitated by the participation of the immune system in countering this growth.  More recent evidence has indicated, however, that quiescent single disseminated cancer cells (DCCs) are involved.  An explanation for this quiescence has been elusive: although involvement of the immune system is suspected.  Of course, the question remains that if the immune system is involved why is it not able to eliminate these DCCs entirely.
Douglas T. Fearon and his colleagues from the John Hopkins University School of Medicine studied the role of adaptive immunity in response to DCCs using the mouse model.  Both mice and humans with pancreatic duct adenocarcinoma (PDA) show DCCs resident in liver.  in both cases, these cells display unusual phenotypic characteristics – negative for cytokeratin (CK) 19 and major histocompatibility complex class I (MHCI).

According to the authors, “The absence of MHCI and the occurrence of specific CD8+ T cells in the genetically engineered mouse model of PDA, and possible in patients with PDA, suggested that DCCs may be selected by an anticancer immune response during the metastatic process.” This rationale is represented by the image below.

The lack of the expression of MHCI in DCCs is indicative of Endoplasmic Reticulum (ER) stress.  ER stress occurs within cells in certain pathological conditions when there is an accumulation of unfolded proteins.  Many proteins vital to cell viability are maintained in precise folded configurations.  If the mechanism responsible for maintaining proteins in the folded state is disrupted, this results in so-called ER stress.  In this model, quiescent DCCs lacking the expression of MHCI elude destruction by the CD8+ T cells.  These surviving DCCs can then grow out into full blown metastases if the immune response is subsequently disrupted.  In other words, it is the immune response that selects for quiescent DCCs.  To test this hypothesis, the investigators used a mouse model that would allow them to introduce immunogenic PDA cells into seeded mice livers that were pre-immunized and contained only quiescent DCCs lacking MHC1 and CK19.  Those recipients that were not pre-immunized developed macro-metastases.   As a result, a subpopulation of PDA with the phenotypic characteristics of DCCs were found in vitro and those cells proved to be the precursors of DCCs in vivo.

The authors of this study conclude that, “A PDA-specific adaptive immune response selects DCCs, in which the ER stress response accounts for both quiescence and resistance to immune elimination. Accordingly, outgrowth of DCCs to macro-metastases requires not only relief from the cancer cell–autonomous ER stress response, but also suppression of systemic immunity. Thus, the ER stress response is a cell-autonomous reaction that enables DCCs to escape immunity and establish latent metastases.”

This finding may prove important in developing more effect therapeutic strategies for combating pancreatic cancer that currently has an unacceptably high mortality rate.  

Saturday, June 23, 2018

Somatic Mosaicism

A central concept in modern biology has been that in any individual, all cells contain identical copies of the DNA that establishes the phenotype of that individual.  This assumption seemed so critical to the life of the organism that it has never been routinely tested.  Current research challenges this assumption.

There is a condition referred to as Long QT Syndrome (LQTS).  The genetic form of this disease can result in life-threatening arrythmias of the heart.  In spite of the genetic correlation, only 30 percent of the patients studied tested positive for the genetic markers associated with LQTS.
Stephen R. Quake and his colleagues at Stanford University performed a detailed mosaic DNA analysis of an infant with perinatal LQTS.  As a result of this analysis, it was discovered that 8% of the heart cells were capable of arrythmia.   To further study this intriguing result, 7500 patients presenting with LQTS were studied for mosaicism.

Somatic mosaicism represents the occurrence and subsequent proliferation of DNA variants in cell lineages resulting from differentiation after fertilization.  It is now believed that this mosaicism may play a causal role in a variety of human ailments.  Furthermore, in the case of LQTS, a variant in the sodium channel protein NA 1.5 that is encoded by the SCN5A gene located on chromosome 3 may be the causative factor.

According to the authors, “One report suggests that 6.5% of de novo mutations presumed to be germline in origin may instead have arisen from postzygotic mosaic mutation events, and recent genetic investigations directly interrogating diseased tissues in brain malformations, breast cancer, and atrial fibrillation have revealed postzygotic causal mutations absent from germline DNA. Pathogenic mosaic structural variation is also detectable in children with neurodevelopmental disorders. However, a consequential category of genetic variation has not been surveyed systematically in clinical or research studies of other human diseases.”
These surprising results not only challenge the accepted assumption regarding the homogeneity of genetic information in every cell of the individual, it also may have profound implications for the etiology and treatment of human disease.

Saturday, May 26, 2018

Impact of the Changing Climate on Rio Grande

The current state of the Rio Grande as reported in the New York Times

Friday, May 11, 2018

The FOXG1 Syndrome

The so-called “FOXG1 Syndrome,” is a rare birth defect in which the infant who presents with this condition is abnormally small at birth and during the early stage of neuronal development, the head grows slowly; this eventually leads to an unusually small head in early childhood – a condition referred to as microcephaly.  Upon close examination there are number of brain anomalies that are present in patients with this condition –
  • thin or underdeveloped corpus collosum that connects the left and right hemispheres of the brain
  • reduction in the number of anatomical folds (gyri) that are present on the brain’s surface
  • reduced amount of white matter in the brain.  White matter is composed of the Axon bodies of the neurons (see diagram below) that connect the cell bodies of the neurons that make up the so-called grey matter.

Structure of the  Human Neuron

On account of the profound anomalies in brain structure associated with this syndrome, affected individuals present with severe and wide-ranging intellectual disabilities.  These can include involuntary muscular movements, difficulties walking, sleep disturbances, seizures and general irritability.  In addition, abilities in speech and language are often seriously impaired adversely affecting social interactions.  The FOXG1 syndrome has been grouped as an autistic-like disorder.  Although the range of impairments evident closely mirrors that of Rett syndrome, it is now classified as a distinct disease.

In terms of the etiology the FOXG1 syndrome, it is believed to be caused by a mutation in the FOXG1 gene.  The normal gene encodes for a the Forkhead Box G1 protein (see diagram below).  this protein seems to play an essential role in brain development where it functions as a transcription factor that normally represses the activity of certain genes when they are not required.  Extensive research has demonstrated that the FOXG1 gene product is involved in the development of the brain in early embryonic life.  The particular site of action of this transcription factor is the telencephalon that ultimately develops into the cerebrum that is involved in so many critical high-order functions in the human brain including language, sensory perception, learning and memory.

Artistic Rendering of the Forkhead Box G1 Protein

Finally, Rett syndrome is also believed to be caused by a different mutation in the FOXG1 gene leading to a dysfunctional Forkhead Box G1 protein.  Since the origin of these diseases are genetic in origin and impose their deleterious effects during embryonic development, a cure may ultimately reside in the introduction of the normal FOXG1 gene in utero.  This may indeed become a reality at some future time.