Exam 3: Biology

Passage Beta-thalassemia is a blood disorder caused by reduced production of the β-globin subunit of hemoglobin.  The erythrocytes in patients with beta-thalassemia are abnormally small, lack sufficient functional hemoglobin, and are decreased in number.  These individuals require lifelong blood transfusions to correct their erythrocyte shortage.  Each unit of blood transfused contains many milligrams of iron and, consequently, blood iron levels increase due to repeated transfusions.  The excess iron is then continuously deposited in the organs, leading to organ damage.Transferrin (Tf) is a protein that binds free iron in the blood and plays a key role in erythropoiesis, which requires uptake of iron from the blood in a process facilitated by Tf and its cell surface receptor (TfR).  Iron-bound Tf, known as holotransferrin (holoTf), binds TfR to form a complex that is brought into the developing erythrocyte in an endosome.  Acidification of the endosome triggers a conformational change in holoTf, which allows the release of free iron into the cell and the recycling of the remaining complex components to the cell surface.  Non-iron-bound Tf is referred to as apotransferrin (apoTf), and due to its iron-binding capability, researchers believe that apoTf injections may help prevent deposition of excess iron in the organs of patients with beta-thalassemia.To investigate the effect of apoTf administration in a mouse model of beta-thalassemia, mice homozygous for an inactivating deletion in the murine β-globin gene (Hbb^th−3/Hbb^th−3) were given blood transfusions followed by apoTf injection.  Measurements of non-transferrin-bound iron in the blood (NTBI), plasma holoTf, and urinary holoTf were taken immediately and at 15 days post-injection.  Results revealed that NTBI decreased over the tested period.  Experimenters hypothesized that apoTf mediated certain regulatory changes that reduced NTBI in Hbb^th−3/Hbb^th−3 mice by increasing urinary holoTf excretion. -Iron buildup in the liver can lead to scarring and loss of liver function.  If the change in holoTf excretion rate were to cause this form of liver damage, the rate at which holoTf is filtered from the glomerular capillaries must be:

Passage Eukaryotic organelles such as mitochondria and chloroplasts are believed to be relics of formerly free-living prokaryotes.  The transition from a hypoxic (low O₂) to an oxic atmosphere (21% O₂) is said to have enabled primitive eukaryotic anaerobes to engulf ancient aerobic prokaryotes and consequently acquire the ability to produce energy through oxidative phosphorylation.  This endosymbiotic theory of eukaryotic evolution also postulates that endosymbiosis resulted in larger eukaryotic genomes, which originated from the partial transfer of mitochondrial genes to the nuclear genome.  On integration into the host genome, mitochondria-derived genes became indistinguishable from the original nuclear genes.Researchers have alternatively proposed that after a prolonged period of symbiosis, there is a possibility of gene transfer from eukaryotes to prokaryotes.  This hypothesis was initially supported when copper/zinc (Cu/Zn) superoxide dismutase (SOD), a metalloprotein confined to the cytosol of eukaryotic cells, was found in Photobacterium leiognathi.  The free-living bacterium P. leiognathi is also a known symbiont of ponyfish, a small fish species native to the Indian and Pacific Oceans.SODs are antioxidant enzymes that serve as the cell's first line of defense against reactive oxygen species (ROS).  ROS produced by the electron transport chain damage proteins by oxidizing amino acid residues and metal ions on prosthetic groups, but can accumulate during times of biochemical and environmental stress.  Superoxide (O₂⁻) radicals, a form of ROS, are sequestered by SODs and converted into less toxic hydrogen peroxide (H₂O₂) and O₂ gas. Adapted from Bannister JV, Parker MW. The presence of a copper/zinc superoxide dismutase in the bacterium Photobacterium leiognathi: a likely case of gene transfer from eukaryotes to prokaryotes. Proc Natl Acad Sci USA. 1985;82(1):149-52. -Based on the passage, which of the following best explains how mitochondrial genes differed from eukaryotic genes prior to gene transfer?  Mitochondrial genes were:

Passage Parathyroid hormone (PTH) is an important regulator of calcium and phosphate homeostasis.  Specifically, PTH functions to increase plasma calcium concentration and reduce plasma phosphate levels.  PTH secretion is closely regulated by changes in plasma calcium concentration, which are detected by the human calcium-sensing receptor (hCaSR), a G protein-coupled receptor located on chief cells of the parathyroid gland.  hCaSR activation ultimately inhibits PTH release by inducing a second messenger cascade in which intracellular calcium levels rise due to the hydrolysis of the membrane phospholipid phosphatidylinositol bisphosphate (PIP₂) into inositol trisphosphate (IP₃) and diacylglycerol (DAG) by the membrane-bound enzyme phospholipase C (PLC).Secondary hyperparathyroidism is a condition characterized by excessive PTH release in response to depressed serum calcium or elevated serum phosphate levels.  The novel peptide AMG 416, an hCaSR agonist, is being investigated as a possible treatment for secondary hyperparathyroidism caused by end-stage renal disease (ESRD).  IP-1, a downstream metabolite of IP₃, is a marker of hCaSR activity.  Researchers used embryonic kidney cells lacking hCaSR (HEK-293T) and HEK-293T clone cells stably transfected with hCaSR (hCaSR) to measure IP-1 levels following AMG 416 treatment (Figure 1). Figure 1  IP-1 concentration in embryonic kidney cells lacking hCaSR (HEK-293T) and HEK-293T clone cells transfected with hCaSR (hCaSR) following AMG 416 treatmentThe dependence of AMG 416 on extracellular calcium was investigated by incubating hCaSR cells with increasing concentrations of AMG 416 in the presence or absence of calcium treatment (Figure 2). Figure 2  IP-1 levels in hCaSR cells incubated with and without calcium and AMG 416 peptide  (Note:  IP-1 levels are normalized to the number of cells present in each culture.)In addition, PTH concentration was measured in 5/6 Nx mice, an animal model of secondary hyperparathyroidism in human ESRD.  Blood samples were taken from male and female 5/6 Nx mice for 8 hours following administration of varying concentrations of AMG 416 (Figure 3), and PTH levels were measured. Figure 3  Serum PTH concentration following administration of AMG 416 to 5/6 Nx mice -Assume that 5/6 Nx mice have a normal physiological response to changes in PTH secretion, and that AMG 416 treatment suppresses PTH release.  Which graphic represents the blood phosphate levels that would be expected in 5/6 Nx mice eight hours after injection with indicated dosages of AMG 416?

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. -The germ layer that gives rise to the notochord also gives rise to which of the following cell types?

A single diploid cell containing 46 chromosomes underwent meiosis and produced four daughter cells, two with 22 chromosomes and two with 24 chromosomes.  An error in which of the following stages of meiosis would most likely explain this result?

Passage The Namibian cheetah (Acinonyx jubatus jubatus), the world's fastest land-dwelling animal, hunts across the African grasslands using high-speed sprints.  Cheetahs possess unique anatomical features specialized for speed, including an aerodynamic skull, elongated legs, and an enlarged heart.  These physiological adaptations have occurred over time and allow cheetahs to achieve speeds greater than 100 km/h.  However, due to their small body and slender limbs, cheetahs (50-64 kg) are limited with regard to the size of the animals they can successfully hunt and therefore typically prey on slightly smaller species that inhabit open grasslands.It is believed that around 12,000 years ago, Namibian cheetahs experienced an environmental catastrophe that drastically reduced their population.  The small number of surviving cheetahs began mating with one another, which led to severe inbreeding depression.  Over time, the offspring of subsequent generations exhibited decreased fitness, reduced fecundity (number of offspring), and sustained a loss of major histocompatibility complex (MHC) allele diversity.  To study if low MHC diversity correlates with low fitness in cheetahs, scientists analyzed the genetic diversity and disease prevalence in a group of free-ranging and captive male cheetahs.Experiment 1MHC allele genotyping and analysis was performed in wild (free-ranging) and captive male cheetahs (Figure 1).  Because MHC diversity is strongly correlated with genetic diversity, MHC diversity is used as a marker for an organism's overall genetic variability. Figure 1  Evaluation of heterozygosity in (A) wild and (B) captive male cheetahs born between 1976 and 2007 (Note:  Each data point represents a single male cheetah.)Experiment 2Disease prevalence was evaluated in wild and captive male cheetahs by quantitative analysis of symptoms relating gastrointestinal, liver, and kidney disease.  The percentages of cheetahs affected by each disease are shown in Figure 2. Figure 2  Evaluation of disease prevalence in wild and captive male cheetahsExperiment 3Fecal cortisol levels were noninvasively evaluated in wild and captive cheetahs over a period of 6 months (Figure 3). Figure 3  Average fecal cortisol concentrations of wild and captive male cheetahs Adapted from Castro-prieto A, Wachter B, Sommer S. Cheetah paradigm revisited: MHC diversity in the world's largest free-ranging population. Mol Biol Evol. 2011;28(4):1455-68. -Assume that a subgroup of Namibian cheetahs exclusively hunt prey that weigh more than 100 kg.  The individuals that make up this subgroup possess larger claws and teeth compared to average Namibian cheetahs and retain the ability to naturally mate with them.  These morphological and behavioral changes reflect:

Passage The lac operon encodes the proteins that metabolize lactose and is a key biochemical feature that differentiates between members of the Enterobacteriaceae family.  For example, the severely virulent Salmonella typhimurium can be distinguished from its less virulent relative Escherichia coli by the absence of a lac operon.  Researchers propose that the evolutionary loss of the lac operon may contribute to S. typhimurium's ability to invade epithelial cells.To test the hypothesis, wild-type (WT) S. typhimurium cells were transformed with plasmids encoding components of the lac operon (Figure 1): β-galactosidase (lacZ⁺), lactose permease (lacY⁺), transacetylase (lacA⁺), or all three (lac⁺).  Each plasmid also contained genes for the lac repressor (lacI), which can inhibit lac operon transcription in the absence of lactose; the cAMP receptor protein (crp), which induces lac expression in low glucose; and a chloramphenicol resistance gene (cat). Figure 1  Plasmid PYM-LACZYA (lac⁺), used to transform WT S. typhimurium.  Similar plasmids containing only one of the three lacZ, lacY, or lacA genes were also used.Virulence assays were performed in mice by inoculating with WT or lac⁺ S. typhimurium.  The assays confirmed that lac⁺ bacteria were less virulent than their WT counterparts, and that both WT and lac⁺ bacteria were more virulent in the presence of glucose.  Subsequently, several genes related to invasion were analyzed for their expression levels in the presence or absence of lac plasmids (Table 1; Figure 2).Table 1  Transcription Levels of Invasion-Associated Genes in lac⁺ S. typhimurium Relative to WT Figure 2  Transcript concentrations of two S. typhimurium flagellar genes, fljB and fliC, in bacteria transformed with lacZ⁺, lacY⁺, lacA⁺, or lac⁺ plasmids.  Concentrations are expressed as a percentage of WT levels. Adapted from Jiang L, Ni Z, Wang L, Feng L, Liu B. Loss of the lac operon contributes to Salmonella invasion of epithelial cells through derepression of flagellar synthesis. Curr Microbiol. 2015;70(3):315-23. -Three antibiotic disks were placed on a bacterial culture.  One disk contained gentamicin (G), one contained chloramphenicol (C), and one contained ampicillin (A).  Based on this information, which of the following cultures represents a strain of Salmonella that has been transformed with the plasmid shown in Figure 1?

Passage Alzheimer disease (AD) is a progressive condition affecting memory and cognition that can be pathologically characterized by the presence of neurofibrillary tangles (NFTs).  NFTs, aggregates of hyperphosphorylated tau proteins that have dissociated from microtubules, are believed to contribute to neuronal degradation and subsequent AD symptoms.  In general, ribosomes initiate translation of tau mRNA by binding the 7-methylguanosine (m7Gpp) cap present at the end of the 5′ untranslated region (UTR) of the mRNA molecule.  However, researchers believe that tau protein translation initiation can occur without recognition of the m7Gpp cap.  This alternate mechanism, referred to as cap-independent translation, indicates that ribosomes are recruited by internal ribosome entry sites (IRES) generally located in the 5′ UTR of the mRNA sequence.To determine the initiation mechanism of tau mRNA translation, neuroblastoma cells of AD mice were transfected with two different DNA constructs (Figure 1).  Construct 1 contained the 5′ UTR of tau mRNA isoforms (v1 and v2) extracted from cells displaying NFTs.  Construct 2 included the 5′ UTR of the β-globulin gene, which normally codes for a plasma protein.  The luciferase reporter genes, Renillla and Photinus, were also included in the two constructs. /_ /_ Figure 1  DNA constructs containing β-globulin or tau gene used to transfect neuroblastoma cellsLuciferase gene expression from the constructs was monitored in the absence and presence of a synthetic m7Gpp cap analog (Figure 2).  Translation of Renillla luciferase mRNA depended on ribosomal binding to the m7Gpp cap of the mRNA, but translation of Photinus luciferase required ribosome recruitment by an IRES in the 5′ UTR of the adjacent gene. Figure 2  Luciferase activity in neuroblastoma cells exposed to m7Gpp cap analogIn a second study, researchers used siRNA to target initiation factor eIF4E, a peptide translated in a cap-dependent manner that facilitates the binding of ribosomes to the m7Gpp cap.  Expression of factor eIF4E, elongation factor eEF2K, and tau v2 protein was assessed in siRNA-treated cells (Figure 3). Figure 3  Western blot of eIF4E, eEF2K, and tau v2 extracted from non-AD cells treated with eIF4E siRNA Adapted from Veo BL, Krushel LA. Translation initiation of the human tau mRNA through an internal ribosomal entry site. J Alzheimers Dis. 2009; 16(2):271-5. -The translation of tau mRNA would most likely involve:an enzyme with the ability to hydrolyze peptide bonds.tRNA molecules carrying amino acids corresponding to codons on the mRNA molecule.a ribosomal complex capable of reading the target mRNA in a 3′ to 5′ direction.

Passage Hematological anomalies have been observed in patients with prolonged liver damage.  For example, patients with chronic cirrhosis (scarring of the liver) often develop anemia and other blood disorders.  Hemoglobin is the metalloprotein within red blood cells that is responsible for the oxygen-carrying capacity of blood, and its function may be affected by liver damage.  The poor prognosis of patients with cirrhosis and anemia has stimulated further study of hemoglobin function and oxygen saturation.Blood samples from both cirrhotic patients and healthy individuals at rest and after exercise were exposed to varying partial pressures of oxygen.  Measurement of oxyhemoglobin dissociation curves (ODCs [Figure 1]) from the blood samples revealed significant functional differences in the hemoglobin from cirrhotic patients relative to control subjects. Figure 1  Oxyhemoglobin dissociation curve for cirrhotic patients (CP) at rest and for healthy controls (HC) at rest and after 20 minutes of exerciseVitamins are considered nontoxic supplements and are frequently provided to anemic patients to increase red blood cell production.  They may therefore have therapeutic benefits for cirrhotic patients.  However, some studies indicate that excessive vitamin consumption may lead to oxidative stress that could induce anemia through hemolysis (red blood cell rupturing).  Hemoglobin strongly absorbs light at 577 nm, and researchers conducted hemolysis assays by spinning down red blood cells and measuring absorbance (Abs) of the supernatant at 577 nm to determine how much hemoglobin was released.  The assay was carried out in the presence of four vitamins to determine their potential as oxidizing agents, and results are shown in Table 1.Table 1  Hemolysis of Red Blood Cells from Healthy Donors Treated With Vitamins [2 mM] for 30 Minutes, Measured by Absorbance at 577 nm Adapted from Clerbaux T, Detry B, Geubel A, et al. The oxyhemoglobin dissociation curve in liver cirrhosis. Chest. 2006;129(2):438-45. -Healthy subjects show a shallow increase in oxygen-carrying capacity at 0-20 mm Hg, but a sharp increase at 20-50 mm Hg.  Which statements explain this effect?Oxygen binding induces a change from the R state to the T stateThe affinity of hemoglobin for oxygen increases with O₂ pressureHemoglobin exhibits positive binding cooperativityHemoglobin exhibits negative binding cooperativity

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. -When expressed, the most likely function of the products generated by the miR-17~92 gene cluster is to:

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 -The information in the passage suggests that increased activity of RANK is most likely due to signaling involving which of the following hormones?

Passage In the central nervous system, myelin produced by oligodendrocytes (glial cells) functions as an insulating sheath surrounding certain nerve fibers.  To identify candidate genes involved in the myelination process, researchers collected oligodendrocytes from zebrafish and purified the messenger RNA (mRNA) transcripts expressed by these glial cells.  The mRNA was converted to complementary DNA (cDNA) by reverse transcriptase, and the cDNA was fluorescently labeled and assessed for hybridization on a microarray.  The expression of mRNA in other zebrafish cells was similarly measured.Transcripts detected in zebrafish oligodendrocytes at levels greater than 3 times those found in other cells were selected as candidate myelination genes.  Some of the detected genes, including plp1a, Sox10, and mbp, were previously known to be specific to oligodendrocytes, validating the procedure.  The cDNA of newly identified candidates was amplified by PCR, and the ends were digested with the restriction enzymes EcoRI and XhoI.  Genes were then ligated into the multiple cloning site (MCS) of the pSKII vector, shown in Figures 1 and 2, which had also been digested by EcoRI and XhoI. Figure 1  Overview of the pSKII vector (plasmid) Figure 2  The pSKII MCS, showing the restriction sites of several restriction enzymesTable 1 shows the functional parameters of several restriction enzymes. To confirm that the candidate genes originated in oligodendrocytes, complementary RNA (cRNA) strands were synthesized from the cloned plasmids and hybridized to fixed zebrafish sections.  One of the genes that localized to oligodendrocytes, known as cldnk, produces a 915 base pair mRNA transcript from two exons.  The cldnk gene is involved in tight junction formation and may be required for myelin sheath development around axons. -Which experiment could confirm that the cldnk gene product is required for myelination?

Passage Eukaryotic organelles such as mitochondria and chloroplasts are believed to be relics of formerly free-living prokaryotes.  The transition from a hypoxic (low O₂) to an oxic atmosphere (21% O₂) is said to have enabled primitive eukaryotic anaerobes to engulf ancient aerobic prokaryotes and consequently acquire the ability to produce energy through oxidative phosphorylation.  This endosymbiotic theory of eukaryotic evolution also postulates that endosymbiosis resulted in larger eukaryotic genomes, which originated from the partial transfer of mitochondrial genes to the nuclear genome.  On integration into the host genome, mitochondria-derived genes became indistinguishable from the original nuclear genes.Researchers have alternatively proposed that after a prolonged period of symbiosis, there is a possibility of gene transfer from eukaryotes to prokaryotes.  This hypothesis was initially supported when copper/zinc (Cu/Zn) superoxide dismutase (SOD), a metalloprotein confined to the cytosol of eukaryotic cells, was found in Photobacterium leiognathi.  The free-living bacterium P. leiognathi is also a known symbiont of ponyfish, a small fish species native to the Indian and Pacific Oceans.SODs are antioxidant enzymes that serve as the cell's first line of defense against reactive oxygen species (ROS).  ROS produced by the electron transport chain damage proteins by oxidizing amino acid residues and metal ions on prosthetic groups, but can accumulate during times of biochemical and environmental stress.  Superoxide (O₂⁻) radicals, a form of ROS, are sequestered by SODs and converted into less toxic hydrogen peroxide (H₂O₂) and O₂ gas. Adapted from Bannister JV, Parker MW. The presence of a copper/zinc superoxide dismutase in the bacterium Photobacterium leiognathi: a likely case of gene transfer from eukaryotes to prokaryotes. Proc Natl Acad Sci USA. 1985;82(1):149-52. -Ponyfish cells containing P. leiognathi symbionts were exposed to a spindle fiber toxin that inhibits microtubule polymerization.  Given this information, which of the following would most likely result as a consequence of toxin exposure?

Passage Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a bacterium responsible for causing foodborne illnesses.  EHEC virulence, or the ability to cause disease, is acquired through quorum sensing, a form of bacterial cell-cell communication dependent on population density.  EHEC O157:H7 bacteria monitor population density by detecting changes in the concentration of autoinducer-3 (AI-3), an extracellular signaling molecule secreted by many bacterial species.  AI-3 generally acts on neighboring bacteria to activate the genes on the enterocyte effacement (LEE) locus.  These genes code for type III secretion proteins (EspA, EspB, EspD, Tir), which promote entry of Shiga-like toxins secreted by EHEC into host intestinal epithelial cells.  This ultimately leads to cell death, lesions, and symptoms such as bloody diarrhea and abdominal cramps.It has been reported that a mutation in S-ribosylhomocysteine lyase (LuxS), an enzyme involved in quorum sensing, may result in diminished production of AI-3.  However, researchers predict that certain peptide hormones can activate LEE gene transcription in the LuxS.  To test this hypothesis, mutant EHEC bacteria deficient in LuxS (LuxS⁻) were cultured and separately exposed to the hormones epinephrine (E), norepinephrine (NE), gastrin (G), and secretin (S) at 37°C.  Total RNA was extracted from the treated bacterial cultures, and LEE mRNA was quantified (Figure 1). Figure 1  Concentration of LEE mRNA in mutant LuxS⁻ bacteria supplemented with purified peptide hormones (Note:  C = control.)Western blot was performed to compare the presence and concentration of type III secretion proteins from wild-type (WT), LuxS⁻, and LuxS⁻ cultured in preconditioned media (PCM).  PCM was generated by culturing uninfected intestinal epithelial cells in growth media for 24 hours.  After 24 hours, the cells were removed and the PCM was used to supplement the growth of LuxS⁻ bacteria. Figure 2  Western blot results of type III secretion proteins isolated from WT, mutant LuxS⁻, and LuxS⁻ in PCM -Which proteins identified in Figure 2 have the shortest amino acid sequence and the greatest concentration in WT cells, respectively?

Passage The Namibian cheetah (Acinonyx jubatus jubatus), the world's fastest land-dwelling animal, hunts across the African grasslands using high-speed sprints.  Cheetahs possess unique anatomical features specialized for speed, including an aerodynamic skull, elongated legs, and an enlarged heart.  These physiological adaptations have occurred over time and allow cheetahs to achieve speeds greater than 100 km/h.  However, due to their small body and slender limbs, cheetahs (50-64 kg) are limited with regard to the size of the animals they can successfully hunt and therefore typically prey on slightly smaller species that inhabit open grasslands.It is believed that around 12,000 years ago, Namibian cheetahs experienced an environmental catastrophe that drastically reduced their population.  The small number of surviving cheetahs began mating with one another, which led to severe inbreeding depression.  Over time, the offspring of subsequent generations exhibited decreased fitness, reduced fecundity (number of offspring), and sustained a loss of major histocompatibility complex (MHC) allele diversity.  To study if low MHC diversity correlates with low fitness in cheetahs, scientists analyzed the genetic diversity and disease prevalence in a group of free-ranging and captive male cheetahs.Experiment 1MHC allele genotyping and analysis was performed in wild (free-ranging) and captive male cheetahs (Figure 1).  Because MHC diversity is strongly correlated with genetic diversity, MHC diversity is used as a marker for an organism's overall genetic variability. Figure 1  Evaluation of heterozygosity in (A) wild and (B) captive male cheetahs born between 1976 and 2007 (Note:  Each data point represents a single male cheetah.)Experiment 2Disease prevalence was evaluated in wild and captive male cheetahs by quantitative analysis of symptoms relating gastrointestinal, liver, and kidney disease.  The percentages of cheetahs affected by each disease are shown in Figure 2. Figure 2  Evaluation of disease prevalence in wild and captive male cheetahsExperiment 3Fecal cortisol levels were noninvasively evaluated in wild and captive cheetahs over a period of 6 months (Figure 3). Figure 3  Average fecal cortisol concentrations of wild and captive male cheetahs Adapted from Castro-prieto A, Wachter B, Sommer S. Cheetah paradigm revisited: MHC diversity in the world's largest free-ranging population. Mol Biol Evol. 2011;28(4):1455-68. -Which of the following statements best explains the trend shown in Figure 1?  (Note:  Captive cheetahs are generally housed individually.)

A student analyzing actively dividing cells under the microscope finds that each daughter cell contains half the number of chromosomes as the parent cell.  Given this, these cells most likely contain all of the following EXCEPT:

Researchers are studying a single-celled pathogen.  Which experimental method would be LEAST effective for determining whether the pathogen is prokaryotic or eukaryotic?

Passage Skin grafting is a procedure in which healthy skin from a different region of the body is transplanted onto an area of absent or damaged skin.  Skin grafts are used for partial-thickness burns, which damage the epidermis and dermis, and for full-thickness burns, which damage the epidermis, dermis, and hypodermis.  Superficial burns injure only epidermal tissue and heal without skin grafts.  Skin grafting has greatly improved the long-term survival of burn victims.  However, because grafted skin cannot effectively dissipate heat, burn survivors are at an increased risk of hyperthermia (abnormally high body temperature), a condition that can lead to heat-related medical emergencies such as heat stroke.  The greater the grafted body surface area (BSA), the greater the risk for complications related to thermoregulation.Heat acclimation (HA) is the process by which the body increases heat tolerance through adaptive changes in the sympathetic control of skin heat dissipation.  It can be induced in healthy individuals by repeated exercise at elevated ambient temperature and humidity.  A group of researchers tested whether skin graft recipients could also undergo HA.  Skin graft recipients who were otherwise healthy and had at least 15% grafted BSA were recruited for the study, as were controls with no history of receiving grafts.  The skin graft recipients were split into two groups, those with 15%-40% grafted BSA and those with >40% grafted BSA.  For a period of seven days, subjects participated daily in 90 min of exercise at 50% maximal oxygen consumption in a room where the temperature was maintained at 40°C.  Exercise modality, fluid intake, and frequency and duration of rest were strictly monitored.On the days immediately preceding and following the HA regimen, the heat tolerance of participants was tested with a 90-min exercise regimen at a fixed heat production rate (Figure 1).  Tolerance testing was halted before 90 min for subjects whose internal body temperature exceeded 40°C.  The change in internal temperature of participants was calculated by subtracting their internal temperature before exercise from their internal temperature at exercise cessation (either once temperature reached 40°C or at the 90-min endpoint). Figure 1  HA in controls and in skin graft recipients Adapted from Schlader ZJ, Ganio MS, Pearson J, et al. J Appl Physiol. 2015;119(1):69-76. -Given that scar tissue forms when collagen-producing cells replace normal tissue after an injury, all of the following processes would be impaired in the area of scarred skin that forms over a partial-thickness burn EXCEPT:

Passage The fruit fly Drosophila melanogaster is frequently used as a model system to investigate neurological disorders.  The genes Abl, Enabled (Ena), and receptor protein tyrosine phosphatase (PTP) are involved in axonal guidance during neuronal development and are all believed to operate in the same pathway.  To investigate interactions between PTP and either Ena or Abl, fly lines were developed in which mutations of each gene were easily identifiable by eye characteristics.  In flies that would normally have white eyes, a dominant mutation that inactivates PTP results in red and white eyes; a dominant Ena mutation yields orange eyes (or red and orange if the PTP and Ena mutations are both present); and a dominant Abl mutation yields elongated eyes.  Using these fly lines, the following experiments were conducted.Experiment 1Females heterozygous for PTP (eePp) were crossed with males heterozygous for Ena (Eepp).  F1 generation males and females, each with red and orange eyes, were then selected and crossed with each other to produce an F2 generation.  Flies with two mutant Ena alleles (EE), as well as those with two mutant PTP alleles and at least one mutant Ena allele (EePP), did not survive past the larval stage.Experiment 2PTP and Abl are located on the same chromosome.  Females with the PTP mutation on one copy of the chromosome and the Abl mutation on the other copy were crossed with wild-type males.  In Drosophila, recombination occurs only in female gametes.  Progeny with parental and recombinant genotypes are shown in Table 1.  F1 males with red and white elongated eyes were then crossed with homozygous PTP mutant females.  This produced an F2 generation in which 50% of both male and female progeny had elongated eyes.Table 1  Phenotypes of 500 F1 Flies Like humans, male flies have a single X chromosome, whereas females have two X chromosomes.  Unlike humans, female Drosophila can store sperm and fertilize multiple eggs over the course of several days after a single mating.  Researchers ensured that females were virgins prior to setting up each cross to avoid contamination. Adapted from Song JK, Giniger E, Desai CJ. The receptor protein tyrosine phosphatase PTP69D antagonizes Abl tyrosine kinase to guide axons in Drosophila. Mech Dev. 2008;125(3-4):247-56. -Of the genes tested in the passage, which are autosomal?EnaAblPTP

During muscle contraction, the power stroke is the pivoting motion of the myosin head that pulls the actin filament inward to shorten the sarcomere.  Given this, which of the following events immediately precedes the power stroke?