Exam 3: Biology

Passage Adeno-associated virus 2 (AAV2) is a nonpathogenic, nonenveloped DNA virus that requires helper viruses such as adenovirus (AV) or herpes simplex virus (HSV) to replicate efficiently.  Because it can transduce human cells, including quiescent (nondividing) cells, without causing disease, AAV2 is attractive as a gene therapy vector.  In clinical trials, a modified AAV2 virus has successfully treated the retinal disease Leber's congenital amaurosis as well as the blood clotting disorder hemophilia B.  AAV2 transduces cells via a multistep process, shown in Figure 1. Figure 1  Schematic showing the mechanism of transduction by AAV2Studies indicate that unlike AV and HSV, AAV2 inhibits pathways other than the endosomal pathway, such as trafficking to the Golgi, increasing the percentage of invading virus particles that arrive at the nucleus. Adapted from Xiao PJ, Samulski RJ. Cytoplasmic trafficking, endosomal escape, and perinuclear accumulation of adeno-associated virus type 2 particles are facilitated by microtubule network. J Virol. 2012;86(19):10462-73. -In quiescent cells, early endosomes containing AAV2 would most likely move toward:

Passage Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease affecting 1 in 10,000 live births.  The pathological hallmark of SMA is the loss of α-motor neurons in the spinal cord, which causes progressive paralysis and, ultimately, premature death in infants.  SMA is caused by insufficient levels of survival motor neuron (SMN), a regulatory protein required for the maintenance of the spinal cord nerve cells that control voluntary muscle movement.The SMN protein is generated from the SMN1 and SMN2 genes, which differ in sequence by only a single nucleotide in exon 7.  In healthy individuals, the gene product of SMN1 is the full-length (FL) SMN protein, and the gene products of SMN2 are both the FL-SMN protein as well as a truncated unstable protein (SMNΔ7) that is rapidly degraded.  Approximately 90% of the SMN2 gene product is SMNΔ7, and only 10% is FL-SMN (Figure 1).  In most patients with SMA, SMN1 is mutated such that its protein product is nonfunctional or deleted.  The disease phenotype arises because the SMN2 gene cannot fully compensate for the deficiency in FL-SMN protein synthesis. Figure 1  Structure of SMN1 and SMN2 transcriptsAntisense oligonucleotides (ASOs) that bind pre-mRNA were injected into SMA mice (SMN1^−/−, SMN2^+/+) exhibiting progressive tail necrosis, a characteristic measure of SMA severity.  ASO therapy effectiveness was assessed by measuring the tail length of wild-type (WT) mice and SMA mice exposed to increasing concentrations of ASOs (Figure 2). Figure 2  Tail length progression of WT mice compared to SMA mice treated with either saline or ASOs Adapted from Lorson CL, Hahnen E, Androphy EJ, Wirth B. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci USA. 1999;96(11):6307-11. -The sequence below is a portion of exon 7 of the SMN1 mRNA transcript.5′ - UCAAGUGAUUCUCCU - 3′Which of the following represents the corresponding DNA coding strand sequence for this particular transcript?

Passage Neurulation refers to the early formation of the nervous system in vertebrate embryos and begins when the notochord, a rodlike mesodermal structure, induces neural tube and neural crest cell formation in the ectoderm.  Neural tube defects (NTDs) arise from abnormalities in the development of the neural tube and can range from mild to severe, depending on which portion of the tube is affected.  Spina bifida, a form of NTD, is divided into three categories.  The most serious subclassification of spina bifida is myelomeningocele, which occurs when the lower spine fails to close, which leaves the spinal cord and nerve roots in the lower back damaged and exposed.NTDs are thought to be caused in part by folate deficiency.  Folate plays a critical role in normal cell division and growth as it is a necessary coenzyme for DNA synthesis and methylation.  Mutations in several genes involved in folate metabolism have been associated with an increased risk for NTDs.  It has been hypothesized that folate decreases NTD risk by ameliorating oxidative stress, thereby reducing damage to developing cells in the embryo.  Certain maternal conditions, such as high blood glucose, increase the risk of NTD development by enhancing embryonic oxidative stress during crucial developmental periods.A study was conducted to examine how supplementation with folic acid, the dietary form of folate, affected pregnant women who were identified to have preexisting diabetes, pregestational obesity (another risk factor for NTD), or no preexisting conditions (control group).  Higher folic acid intake decreased the incidence of embryonic NTDs among pregnant women with diabetes, but no significant effect was found for pregnant women who were obese. Adapted from Parker SE, Yazdy MM, Tinker SC, Mitchell AA, Werler MM. The impact of folic acid intake on the association among diabetes mellitus, obesity, and spina bifida. Am J Obstet Gynecol. 2013;209(3):239.e1-8. -Compared to newborns without NTDs, a newborn diagnosed with myelomeningocele would most likely have exhibited which of the following during gestation?

Passage At the blood-brain barrier, the cerebral spinal fluid (CSF) is separated from lymphatic circulation by epithelial cells bound by tight junctions.  Within the central nervous system (CNS), CSF surrounds and protects the brain and spinal cord and is believed to function in waste clearance for the CNS analogous to the role of the lymphatic system within the body.This waste clearance system has been dubbed the "glymphatic system."  Although the mechanism of clearance is largely unknown, specialized glial cells known as astrocytes appear to modify the interstitial volume between neurons.  This increase in interstitial volume allows for greater CSF flow, which increases the efficiency of neurotoxic clearance.  Waste products removed from the brain include metabolic products such as ammonia and harmful compounds such as amyloid proteins.Recent advances in imaging technology suggest that interstitial clearance may be modified during sleep.  To test this hypothesis, researchers indirectly studied changes in interstitial volume by measuring the percentage of CSF coverage in animal models during the first hour of sleep and again during the first hour of wakefulness (Figure 1). Figure 1.  Percentage of CSF coverage of interstitial space during sleep and wakefulness -A patient ingests the following pharmaceutical compound prior to sleep. During the hours following ingestion, the drug concentration in the brain will:

Passage Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic synovial (joint) inflammation.  RA immunopathogenesis involves LFA-1, a surface receptor found on leukocytes that is composed of an α-chain (CD11a) and a β-chain (CD18).  The binding of LFA-1 to ICAM-1, an adhesion protein on the surface of B cells and endothelial cells, stimulates the migration of leukocytes to specific areas of inflammation.  After successful LFA-1/ICAM-1 binding, CD18 is released into the plasma and synovial fluid (SF), where it exists in soluble form (sCD18).  The levels of sCD18 in SF can be used as a marker for inflammation in RA.CD18 release has been shown to increase in response to tumor necrosis factor (TNF), a pro-inflammatory cytokine involved in the inflammatory response of RA.  In patients with newly diagnosed and untreated RA (eRA), leukocytes produce adequate quantities of LFA-1 and plasma sCD18 levels are unchanged.  However, over time there is dysregulation of LFA-1 synthesis and an increase in competition for ICAM-1 by other ligands, leading to a reduction in CD18 shedding.  As a result, sCD18 is decreased in patients with chronic RA (cRA).Experiment 1An immunofluorescence assay was used to measure plasma sCD18 levels in patients with eRA and cRA, and in age- and gender-matched healthy controls (HC). Figure 1  Baseline sCD18 measurements (Note: 50% of data points are below the median value; median = line inside the box.)Experiment 2Clinical assessments were performed in eRA patients to establish a baseline disease status.  eRA patients were assigned DAS28CRP scores measured by RA-specific laboratory values.  This was followed by treatment with anti-inflammatory drugs.  DAS28CRP scores were calculated at 3-month intervals for 12 months, with lower scores indicating decreased RA severity (Figure 2). Figure 2  DAS28CRP scores in eRA patientsExperiment 3Heterogeneous populations of mononuclear cells were cultured from the synovial fluid (SFMC) and peripheral blood (PBMC) of eRA patients during an acute exacerbation of their symptoms.  sCD18 levels were measured in SFMCs and PBMCs that were untreated, treated with TNF, or treated with monoclonal antibodies targeting TNF (anti-TNF). Adapted from Kragstrup TW, Jalilian B, Keller KK, et al. Changes in Soluble CD18 in Murine Autoimmune Arthritis and Rheumatoid Arthritis Reflect Disease Establishment and Treatment Response. PLoS ONE. 2016;11(2):e0148486. -The antibodies used in Experiment 3 are composed of:

Passage Overexpression of the miR-17~92 gene cluster occurs in many human cancers involving c-Myc, a gene coding for a transcription factor that regulates expression of proteins such as p21, an inhibitor of cell cycle progression.  miR-17~92 encodes six distinct microRNAs (miRNAs) that can be divided into four families based on sequence identity at positions 2-7 (Table 1).Table 1  Sequence Identities of miRNA Families Encoded by the mir-17∼92 Gene Cluster Experiment 1Two independent, diffuse B-cell lymphoma cell lines were used to study the functional relationship between miR-17~92 transcripts and c-Myc in cancer pathogenesis.  The first cell line was derived from Eμ-Myc mice (Eμ), which overexpress a c-Myc transgene under the control of a B-cell-specific enhancer that induces early development of B-cell lymphoma.  The second cell line was derived from Eμ-Myc mice carrying a miR-17∼92 knockout allele (Δ/Δ).  As shown in Figure 1, researchers measured the growth of both cell lines, as well as that of Δ/Δ cells infected with a retrovirus expressing the entire miR-17∼92 cluster (Δ/Δ + miR-17∼92). Figure 1  Growth curves of Eμ cells, Δ/Δ cells, and Δ/Δ + miR-17∼92 cells (error bars = standard deviation)Experiment 2To assess the relative contribution of each miRNA to the oncogenic ability of the miR-17∼92 cluster, Δ/Δ cell lines were transduced with an empty vector or with miR-17∼92 mutant allele constructs that were engineered to lack expression of miRNA from at least one of the four families.  In each of the constructs, the family that was knocked out of the vector is indicated by the Δ symbol.  After verifying that the transduced cells correctly expressed the desired miRNA, the fraction of apoptotic cells in each line was measured (Figure 2). Figure 2  Percentage of apoptotic cells in Eμ cells and Δ/Δ cells transduced with the indicated miR-17~92 construct (error bars = standard deviation) Adapted from Mu P, Han YC, Betel D, et al. Genetic dissection of the miR-17~92 cluster of microRNAs in Myc-induced B-cell lymphomas. Genes Dev. 2009;23(24):2806-11. -Based on Figure 2, which family from the miR-17~92 gene cluster is expected to contribute the most to cancer progression?

Passage Bone remodeling is the continuous, microscopic process by which bone tissue throughout the body is resorbed and re-deposited.  Proper regulation of bone remodeling is necessary to maintain bone strength and prevent the onset of conditions of fragile bone such as osteoporosis.  Patients with such conditions have increased fracture risk and generally exhibit decreased activity of osteoblasts (bone-depositing cells), leading to reduced bone density and weakening of the bone matrix.  However, some cases can occur due to overactive osteoclasts (multinucleated cells that mediate bone resorption).  Osteoclasts resorb bone by secreting proteolytic enzymes and acids into the adjacent extracellular space.  These enzymes degrade the organic matrix of bone, and the acid causes the dissolution and release of the mineral components of bone, such as calcium, potassium, and magnesium.  Generally, less than 1% of adult bone is occupied by active osteoclasts.Osteoclast differentiation and activation is thought to be related to the RANK/RANKL signaling pathway.  RANKL, expressed as a membrane-bound or secreted protein, serves as the ligand for RANK, a transmembrane receptor.  In bone tissue, RANK is expressed by precursors of mature osteoclasts, also known as osteoclast progenitor cells (OPCs).  RANKL is expressed by osteoblasts adjacent to OPCs, and the binding of RANKL to RANK in these neighboring cells causes downstream effects through recruitment of TRAF6, an intracellular protein.  TRAF6 activates multiple downstream signaling cascades, but only four of these directly mediate the development of mature osteoclasts from OPCs.  In one of the four pathways, the transcription factor NF-kB is activated, which leads to increased expression of the proto-oncogene c-Fos.  Subsequently, c-Fos interacts with another transcription factor known as NFATc to promote the expression of genes related to osteoclast-specific development and function in OPCs (Figure 1). Figure 1  RANK/RANKL signaling induces differentiation of OPCs into osteoclasts -Based on the passage, the cells that are most important for the differentiation of OPCs are cells that promote:

Passage Neurulation refers to the early formation of the nervous system in vertebrate embryos and begins when the notochord, a rodlike mesodermal structure, induces neural tube and neural crest cell formation in the ectoderm.  Neural tube defects (NTDs) arise from abnormalities in the development of the neural tube and can range from mild to severe, depending on which portion of the tube is affected.  Spina bifida, a form of NTD, is divided into three categories.  The most serious subclassification of spina bifida is myelomeningocele, which occurs when the lower spine fails to close, which leaves the spinal cord and nerve roots in the lower back damaged and exposed.NTDs are thought to be caused in part by folate deficiency.  Folate plays a critical role in normal cell division and growth as it is a necessary coenzyme for DNA synthesis and methylation.  Mutations in several genes involved in folate metabolism have been associated with an increased risk for NTDs.  It has been hypothesized that folate decreases NTD risk by ameliorating oxidative stress, thereby reducing damage to developing cells in the embryo.  Certain maternal conditions, such as high blood glucose, increase the risk of NTD development by enhancing embryonic oxidative stress during crucial developmental periods.A study was conducted to examine how supplementation with folic acid, the dietary form of folate, affected pregnant women who were identified to have preexisting diabetes, pregestational obesity (another risk factor for NTD), or no preexisting conditions (control group).  Higher folic acid intake decreased the incidence of embryonic NTDs among pregnant women with diabetes, but no significant effect was found for pregnant women who were obese. Adapted from Parker SE, Yazdy MM, Tinker SC, Mitchell AA, Werler MM. The impact of folic acid intake on the association among diabetes mellitus, obesity, and spina bifida. Am J Obstet Gynecol. 2013;209(3):239.e1-8. -Assume that neural stem cells are considered to be multipotent.  Which of the following best describes this classification of stem cells?

Passage Quantitative fluorescence recovery after photobleaching (FRAP) is used to study the movement of molecules in live cells.  During FRAP, the fluorescence of a green fluorescent protein (GFP)-tagged molecule is first measured and then photodestroyed in targeted cell regions.  Researchers then assess the time course for fluorescence recovery, which is an indicator of molecular mobility of the GFP-tagged protein into the photodestroyed region.  The average time required to recover 80% of the pre-bleach fluorescence of protein histone 1 (H1) fused to GFP (H1-GFP) in the photodestroyed region is given as t80.FRAP was used to analyze the mobility of the architectural H1 alone and in the presence of high-mobility group (HMG) proteins.  HMG proteins are dynamic modifiers that have been shown to have opposite effects but similar binding sites on chromatin structure. Figure 1  Unique chromatin binding motifs of HMG proteinsDuring analysis, HMGA1 and HMGB1 were microinjected into the cytoplasm of mouse embryonic fibroblast cells expressing H1-GFP.  FRAP was performed on euchromatin and heterochromatin.  Euchromatin and heterochromatin domains were identified as regions weakly and strongly stained by H1-GFP, respectively.  The relative intensity of H1-GFP fluorescence in euchromatin and heterochromatin of uninjected and injected cells was assessed, and t80 results are shown in Table 1.Table 1  Effect of HMGB1 and HMGA1 on H1-GFP Mobility Adapted from Catez F, Yang H, Tracey KJ, Reeves R, Misteli T, Bustin M. Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol Cell Biol. 2004;24(10):4321-8. -Based on Figure 1, HMGN2 is most likely to interact with a histone complex that is rich in:

Which of the following mRNA strands contains the longest open reading frame?

Passage Mitochondria are thought to have been independent bacterial organisms that were engulfed and integrated into eukaryotic cells approximately two billion years ago.  Most of the mitochondrial genome was lost or transferred into the large central genome of the eukaryotic nucleus, leaving only a residual genome within each mitochondrion.Because multiple mitochondria are found in the cell, mitochondrial DNA (mtDNA) mutations result in a condition known as heteroplasmy, or the intracellular mixture of wild-type mtDNA and mutant mtDNA.  Just as the cellular ratio of wild-type to mutant mtDNA varies, the overall mtDNA content within a tissue or organ is also highly variable.  For this reason, disease-causing mutant mtDNA manifests phenotypically only when the majority of mitochondria within a given tissue express the deleterious allele.  In addition, the variability in mtDNA content makes it possible for individuals with the same mitochondrial mutation to display drastically different clinical symptoms.To investigate how heteroplasmy influences disease manifestation, researchers analyzed an A to G substitution at nucleotide 3243 in the tRNA^leu gene of mtDNA.  This mutation has been associated with hereditary hearing loss, diabetes mellitus, and a syndrome characterized by mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes.Family members who were confirmed to have the A3243G mutation were tested using an audiometry examination to assess hearing impairment, as shown in Table 1.  The subjects carried no other genetic mutations known to cause hearing loss.Table 1  Audiometry Results From Family Members with the A3243G Mutation The blood samples of these subjects were collected, and the 3243 region of the tRNA^leu gene was amplified via standard PCR.  The PCR products were subsequently treated with the restriction enzyme ApaI and visualized on a PAGE gel.  The normal undigested PCR product is 161 bp in length, but the PCR product amplified from the mutant A3243G is digested into two fragments by ApaI. Figure 1  PAGE visualization of PCR products following ApaI digestion Adapted from Hadjivasiliou Z, Pomiankowski A, Seymour RM, Lane N. Selection for mitonuclear co-adaptation could favour the evolution of two sexes. Proc Biol Sci. 2012;279(1734):1865-72. -Based on the passage, which of the following reagents was most likely used during PCR?

Passage Obstructive respiratory illnesses are characterized by an increased difficulty exhaling much of the air in the lungs.  Asthma and chronic obstructive pulmonary disease (COPD) are two of the most common obstructive respiratory illnesses.  Asthma is due to allergen hypersensitivity and can be identified by temporary airway inflammation, which causes the bronchioles to narrow.  The decreased airway diameter increases the resistance to airflow.  Subsequently, air becomes "trapped" in the lungs, restricting the amount of air that can be moved in each breath.COPD refers to a spectrum of disorders and can be a result of emphysema, the irreversible destruction of pulmonary tissue.  The breakdown of elastic proteins in the lungs results in a loss of pulmonary resiliency and chronic lung hyperinflation.  Because the walls between individual alveoli are broken down, the alveolar sacs that remain are larger.Lung diseases can be diagnosed and monitored using spirometry.  A spirometer measures the flow rate and volume of inhaled and exhaled air.  In one pulmonary test, a patient is asked to exhale forcibly and completely into a spirometer after maximum inhalation.  This test is done to measure the patient's forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), the total amount of air exhaled in a single breath. Figure 1  Forced expiratory spirogram of a healthy individual Adapted from T. Barreiro, "An Approach to Interpreting Spirometry." American Family Physician. ©2004 AAFP. -During an asthma attack, temporary bronchoconstriction would have what effect on blood pH, and what would be the expected homeostatic response?

One function of endothelial cells is to:

Compared to the blood flowing into the left ventricle, the blood flowing into the right ventricle:

Passage Lipopolysaccharides (LPS) found in gram-negative bacterial cells can elicit a lethal degree of adjuvanticity, or immune activation, in LPS-responsive host organisms.  In mice, responses may be modulated by abnormalities in the toll-like receptor (TLR) encoded by the Tlr4 gene closely linked to coat color on Chromosome 4.  To investigate the effect of a particular Tlr4 mutation on LPS response, three experiments were performed following a cross between fully responsive He-N mice [tlr4(+)/tlr4(+)] and He-J mice homozygous for the mutation [tlr4(d)/tlr4(d)].Experiment 1Researchers isolated spleen cells for analysis of protein expression and LPS response.  In cells isolated from F1 mice, both wild-type and mutant TLRs were distinguishable by protein electrophoresis.  Following this first analysis, spleen cells from each cohort were exposed to 2 × 10⁵ SRC, an antigen formed by sheep erythrocytes.  Cultures were then incubated in the presence or absence of Ph-LPS, with LPS extracted from Escherichia coli K345 cells using a phenol-water mixture.  Titers of anti-SRC antibody were recorded on incubation day 5, with the results shown in Table 1.Table 1  Mean anti-SRC titers with standard error Experiment 2Serum samples were assayed for interferon production 2 hours post LPS injection.  Interferon was quantified using a plaque reduction assay on mouse cells challenged with vesicular stomatitis virus. Figure 1  Average serum interferon levels in mice cohortsExperiment 3Mice were injected with varying doses of Ph-LPS and monitored for 4 days.  The lethal dose (LD₅₀) was defined as the minimum dose causing death in 50% of injected mice.  Compared to He-N mice, results reflected a 10-fold increase in LD₅₀ for F1 mice and a 100-fold increase in LD₅₀ for He-J mice. -Experimental results suggest that the mutated Tlr4 allele exhibits which quality?

Passage Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic synovial (joint) inflammation.  RA immunopathogenesis involves LFA-1, a surface receptor found on leukocytes that is composed of an α-chain (CD11a) and a β-chain (CD18).  The binding of LFA-1 to ICAM-1, an adhesion protein on the surface of B cells and endothelial cells, stimulates the migration of leukocytes to specific areas of inflammation.  After successful LFA-1/ICAM-1 binding, CD18 is released into the plasma and synovial fluid (SF), where it exists in soluble form (sCD18).  The levels of sCD18 in SF can be used as a marker for inflammation in RA.CD18 release has been shown to increase in response to tumor necrosis factor (TNF), a pro-inflammatory cytokine involved in the inflammatory response of RA.  In patients with newly diagnosed and untreated RA (eRA), leukocytes produce adequate quantities of LFA-1 and plasma sCD18 levels are unchanged.  However, over time there is dysregulation of LFA-1 synthesis and an increase in competition for ICAM-1 by other ligands, leading to a reduction in CD18 shedding.  As a result, sCD18 is decreased in patients with chronic RA (cRA).Experiment 1An immunofluorescence assay was used to measure plasma sCD18 levels in patients with eRA and cRA, and in age- and gender-matched healthy controls (HC). Figure 1  Baseline sCD18 measurements (Note: 50% of data points are below the median value; median = line inside the box.)Experiment 2Clinical assessments were performed in eRA patients to establish a baseline disease status.  eRA patients were assigned DAS28CRP scores measured by RA-specific laboratory values.  This was followed by treatment with anti-inflammatory drugs.  DAS28CRP scores were calculated at 3-month intervals for 12 months, with lower scores indicating decreased RA severity (Figure 2). Figure 2  DAS28CRP scores in eRA patientsExperiment 3Heterogeneous populations of mononuclear cells were cultured from the synovial fluid (SFMC) and peripheral blood (PBMC) of eRA patients during an acute exacerbation of their symptoms.  sCD18 levels were measured in SFMCs and PBMCs that were untreated, treated with TNF, or treated with monoclonal antibodies targeting TNF (anti-TNF). Adapted from Kragstrup TW, Jalilian B, Keller KK, et al. Changes in Soluble CD18 in Murine Autoimmune Arthritis and Rheumatoid Arthritis Reflect Disease Establishment and Treatment Response. PLoS ONE. 2016;11(2):e0148486. -Which graphic shows the expected results of Experiment 3?

Passage Within the Actinopterygii (bony fish) lineage, marine teleosts (bony fish with mobile jaws) live in an aquatic environment where there is a higher external concentration of salt and a lower concentration of water relative to the internal concentrations.  Owing to these conditions, salt ions tend to diffuse into the teleost through its skin, whereas water molecules within the organism osmotically traverse the opposite path.Irrespective of lineage, many fish species actively assess their environment and regulate their internal concentration of fluids and electrolytes via a homeostatic process known as osmoregulation.  Teleosts with either glomerular kidneys (eg, eels, sculpin) or aglomerular kidneys that are purely tubular (eg, goosefish, toadfish) have served as experimental subjects for the study of marine teleost osmoregulation.  Analyzing the ionic (ie, Na⁺, Cl⁻, Mg²⁺, SO₄²⁻) concentration of intestinal fluids, urine, and plasma in each species led to the discovery that the renal tubule of the goosefish has both excretory and reabsorptive functions.Scientists have concluded that marine teleost osmoregulation involves the ingestion of seawater, retrieval of NaCl and water from the intestine, and primary excretion of divalent ions via urine and monovalent ions through the gills (thin barriers between the organism's blood and the aquatic environment).In an experiment to further study osmoregulatory mechanisms, researchers emptied the gut of an eel and sealed the anus to prevent fluid loss.  The eel was transported into seawater that had been treated with the volume marker phenol red.  After 20 hours, the eel's gut contained 2.3 mL of fluid but showed a phenol red concentration equivalent to 12.3 mL of ingested seawater.  Researchers found that the eel lost 2.3 g of weight due to urine output.  They ultimately reported that the eel had lost all the seawater it absorbed through the gut via various mechanisms. -Scientists also study freshwater teleosts, which live in an environment where there is more water and less salt outside their bodies than inside.  Osmoregulation in freshwater teleosts would most likely serve to combat their natural tendency to:

Which of the following cellular processes would most likely lead to increased genetic diversity in a population of organisms?

Passage Each time a eukaryotic cell replicates its DNA, the telomeres at the ends of each chromosome become slightly shorter.  When telomeres reach a critically short length, the cells enter the senescent state, in which cell division ceases (Figure 1).  In humans, conditions such as hypertension, liver disease, and Alzheimer disease have been associated with the decreased telomere length and resulting senescence that occur as part of the normal aging process. Figure 1Cells that must be able to divide indefinitely (stem cells and germ cells) express an enzyme known as telomerase.  Functional telomerase consists of a protein subunit called telomerase reverse transcriptase (TERT) and a noncoding RNA subunit called telomerase RNA (TR).  Using a portion of TR as a template, telomerase extends telomeres by continuously adding the sequence 5′-TTAGGG-3′ to the ends of chromosomes.  The complement strand is then filled in by DNA polymerase.  Telomere extension allows these cells to avoid senescence.In healthy somatic cells, however, TERT transcription is generally downregulated by the absence of the oncogene c-Myc and the presence of the tumor suppressor protein WT1.  When telomerase is suppressed, the resulting critical telomere shortening has been hypothesized to prevent uncontrolled cell proliferation in adult tissues.  In cancerous somatic cells, on the other hand, increased telomerase activity is often associated with tumorigenesis.  WT1 is frequently absent and c-Myc is frequently overexpressed in these cells, leading to increased TERT expression.In addition to transcriptional control, TERT can be regulated at the post-transcriptional level.  The TERT gene consists of 16 exons and 15 introns that can be spliced into more than 20 different isoforms in humans.  Only one isoform (the active isoform) is known to extend telomeres.  However, the i2 isoform, in which intron 2 is partially included in the mature mRNA (Figure 2), has been hypothesized to downregulate residual TERT activity in somatic cells. Figure 2 Adapted from Cifuentes-rojas C, Shippen DE. Telomerase regulation. Mutat Res. 2012;730(1-2):20-7. -Considering that healthy somatic cells typically transcribe the TERT gene only at low levels, where in the chromosomes of these cells is the gene most likely found?

Passage A 6-day-old, genotypically normal (46,XX) female patient shows symptoms of hypoglycemia (low blood glucose) and hypotonia (decreased muscle tone).  Physical examination reveals abnormal development of the external genitalia, and laboratory studies show elevated urinary sodium content, increased serum levels of potassium, and abnormal serum concentration of several steroid hormones.  The patient is diagnosed with 21-hydroxylase deficiency, the most common type of congenital adrenal hyperplasia (CAH).CAH refers to a group of autosomal recessive diseases, each associated with defective production of one or more steroid hormones by the adrenal glands, two crescent-shaped glands located above each kidney in the abdominal cavity.  It has been reported that unequal crossing over involving chromosome 6 can result in CAH.  More than 90% of CAH diagnoses are associated with a deficiency of 21-hydroxylase, an enzyme encoded by the CYP21A2 gene on chromosome 6.Normally, 21-hydroxylase catalyzes chemical reactions involved in the synthesis of aldosterone and cortisol, the principal mineralocorticoid and glucocorticoid hormones, respectively.  As a result, patients with 21-hydroxylase deficiency exhibit diminished serum levels of aldosterone and cortisol.  However, synthesis and secretion of the sex hormone testosterone is excessive.  Glucocorticoid deficiency disrupts the negative feedback loop linking the brain to the adrenal gland, leading to hyperplasia (increased cell proliferation) of adrenal tissue.The patient's 21-hydroxylase deficiency is treated with regular administration of synthetic steroid hormones.  Her condition improves after receiving these medications. -What changes in blood volume and serum sodium content would be expected as a result of using synthetic hormones to replenish aldosterone in CAH patients?