Exam 3: Biology

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. -Intraperitoneal injection of insulin would cause the peptide hormone to enter the bloodstream and exert its effect on target cells by:

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. -In vertebrates, researchers found that the concentration pattern of the signaling proteins Sonic Hedgehog (Shh) and Bone Morphogenic Protein (BMP) defined specific regions of expression for various genes involved in neural tube development.  The results of their investigation are shown in the graph provided. Based on the data, which statement best explains how Shh specifies the dorsal and ventral fates of the developing neural tube?

Passage The nicotinic acetylcholine receptor (nAChR) is a protein embedded in the muscle fiber membrane that facilitates cell-to-cell communication across the neuromuscular junction.  When acetylcholine (ACh) binds nAChR, electrical depolarization within skeletal muscle fibers is induced and ultimately gives rise to contractile activity.  The nAChR is composed of five protein subunits symmetrically organized around a central ion pore.  Previous studies demonstrated that these subunits may serve as epitopes (antibody binding sites) in autoimmune disease.  By reversibly binding nAChR subunits, anti-nAChr antibodies may temporarily block the attachment site for ACh, resulting in muscle weakness.Researchers harvested muscle samples from mice to investigate the effects of anti-nAChR antibodies on muscle tension, the pulling force transmitted by muscles during contraction.Experiment 1First, skeletal muscle samples (tibialis anterior) of adult male mice were surgically harvested (n = 30), washed, and stripped of connective tissues.  Next, the isolated muscle samples were placed in a 150-mL oxygen-infused, electrolyte-rich bath containing glucose and Ca²⁺ ions that were able to enter the muscle fibers and enable maximal contractile force.  One end of the submerged muscle was anchored in place while the other was attached to an apparatus that measures muscle tension and modifies muscle length.  Using electrodes, action potentials were elicited in the harvested muscle tissue to evaluate contractile function (Figure 1). Figure 1  Action potential elicited from a harvested skeletal muscle sampleExperiment 2The researchers divided the 30 muscle samples into three groups of 10.  Resting muscle tension was measured, and the electrolyte-rich baths of each group were further infused with a 7.5-mL solution containing mouse-specific anti-nAChR antibodies, ACh, or a mixture of both (Table 1).  Post-infusion (peak) muscle tension was measured for 30 minutes, with results displayed in Figure 2.Table 1  Solutions Infused into the Electrolyte-Rich Bath Figure 2  Pre-infusion resting muscle tension and post-infusion peak muscle tension in harvested muscle samples (Note: * indicates p < 0.05.) -Why was Ca²⁺ added to the oxygen-infused, electrolyte-rich bath?  To allow Ca²⁺ to:

Passage Endometriosis is a chronic, treatable disease of the female reproductive tract associated with infertility and pelvic pain in women of reproductive age.  The endometrium, a layer of epithelial tissue that grows and sheds during the menstrual cycle, normally makes up the interior lining of the uterus.  In individuals with endometriosis, endometrial tissue is also found ectopically (outside the uterine cavity).The mechanism by which endometrial tissue successfully implants outside the uterine cavity is thought to be dependent on signaling molecules that promote the formation of blood vessels locally.  One of these signaling molecules is vascular endothelial growth factor (VEGF), a protein that is normally synthesized and secreted by various tissues in response to reduced oxygen levels.  Because VEGF is thought to be implicated in ectopic endometrial tissue growth, researchers hypothesize that the abdominal and pelvic structures of women with endometriosis would contain higher levels of VEGF compared to women without endometriosis.In an experiment, women ages 25-40 were divided into groups based on endometriosis disease status.  Individuals on any type of medication were excluded from the study.  Using minimally invasive surgery, samples of peritoneal fluid were taken from healthy subjects (n = 19) and from subjects diagnosed with endometriosis (n = 30).  An enzyme-linked immunosorbent assay (ELISA) was used to characterize the concentration of VEGF protein present within these samples.In a separate procedure that took place five days after peritoneal fluid collection, uterine endometrial samples were obtained from all subjects and visualized under a microscope to determine the menstrual cycle phase of each participant.  Figure 1 shows the level of VEGF in the abdominal fluid categorized by disease status and menstrual cycle phase. Figure 1  VEGF concentration in sampled peritoneal fluid (Note:  * = p < 0.05; Proliferative phase = stage immediately preceding ovulation; Secretory phase = stage immediately following ovulation) -Surgical removal of the ovaries is one treatment for endometriosis.  Complete removal of the ovaries would most likely result in which of the following?

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 data in Figure 2 suggest that ribosomes interact with the:

Passage The lumen of the human gut is lined by a monolayer of epithelial cells that acts as a selectively permeable barrier, preventing the passage of harmful intraluminal foreign antigens, flora, and toxins into the circulation while allowing digestion and absorption of essential dietary nutrients along with the transfer of electrolytes and water.Proteins in the tight junctions of intestinal epithelial cells maintain barrier integrity, but barrier dysfunction occurs when these cells are damaged in the setting of infection, burns, shock, or hypoxia (low oxygen levels).  The transcription factor HIF-1, a heterodimer composed of the macromolecules HIF-1α and HIF-1β, regulates the adaptive cellular response to hypoxia and the consequent expression of tight junction proteins.Researchers assessed the concentration of HIF-1 heterodimer components in human intestinal Caco-2 cells subjected to hypoxia/reoxygenation (H/R) in vitro.  Caco-2 cells, a colon-derived cell line, were cultured under specific conditions to mimic the functional and morphological phenotype of wild-type enterocytes lining the small intestine.  These cells were prepared and grown as a monolayer on a collagen-coated membrane.Next, the monolayer was cultured in hypoxic conditions and then exposed to atmospheric oxygen levels (normoxia) for 30, 60, and 120 minutes.  Protein levels were quantified using direct enzyme-linked immunosorbent assay (ELISA), in which an antibody linked to a reporter enzyme was utilized to bind and detect expression of the target molecule (analyte) in a sample.  When the colorless substrate of the reporter enzyme was added, the enzyme generated a visible colored product that could be quantified based on color intensity. Figure 1  Concentration of (A) HIF-1α and (B) HIF-1β in Caco-2 cells subjected to H/R (Note:  0 minutes = cells cultured in hypoxic conditions)Emodin, an anthraquinone compound that prevents hypoxia-induced epithelial cell disruption, was used in conjunction with a chemical HIF-1α inhibitor (HIF-1α-I) to treat Caco-2 cells in a separate experiment.  Transepithelial electrical resistance (TEER) was assessed as a measure of barrier function, with higher TEER values indicating a more intact epithelial cell barrier.  HIF-1α-I was found to block emodin's protective effect on epithelial barrier integrity. Adapted from Lei Q, Qiang F, Chao D, et al. Int J Mol Med. 2014;34(6):1629-39. -A scientist claims that ELISA, although it can accurately detect the concentration of an analyte in a sample, can generate faulty results under certain experimental settings.  Inaccurate quantification of an analyte such as HIF-1α using ELISA would most likely result from all of the following EXCEPT:

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 -Researchers treated 5/6 Nx mice with regular intraperitoneal infusions of CaCl₂ to cause a sustained increase in serum calcium levels.  The hormone that was released as a result of this condition was monitored using a hormone-specific radiolabeled antibody.  In the tested animals, what organ would most likely show increased binding of radiolabeled antibody?

Passage Domestic pigs have been used as valuable preclinical models for scientific research due to their numerous anatomical, developmental, and physiological similarities to humans.  For example, oogenesis and embryonic development in pigs are remarkably similar to the human processes and have facilitated the study of impaired fertilization due to decreased oocyte competence (quality).Researchers have hypothesized that early termination of embryo development can arise from low mitochondrial DNA (mtDNA) copy number.  Because mtDNA replication and reduction events have been extensively mapped in pigs, oocytes and sperm were isolated from their respective porcine gonads to test this hypothesis.Experiment 1To evaluate the developmental competence of porcine oocytes, researchers first measured the activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that is downregulated as the oocyte matures.  The oocytes were stained with Brilliant Cresyl Blue (BCB), a nontoxic blue dye that is converted to a colorless compound by G6PD.  After 90 minutes of staining, oocytes were divided into two groups: one contained oocytes with colorless cytoplasm (BCB⁻) and the other contained oocytes with blue cytoplasm (BCB⁺).  Gene expression profiles were compared to evaluate the number of oocytes in the BCB⁺ and BCB⁻ groups that had developed to metaphase II.Experiment 2BCB⁺ and BCB⁻ oocytes underwent in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or supplementation with homogeneous mitochondria and ICSI (mICSI).  During IVF, an isolated oocyte was placed in a well plate and incubated with sperm at 39 C for 4 hours.  In ICSI, a single sperm was selected and injected directly into the oocyte.Table 1  Developmental Rates for BCB⁺ and BCB⁻ Oocytes Fertilized Subjected to IVF, ICSI, or mICSI Adapted from Cagnone GL, Tsai TS, Makanji Y, et al. Sci Rep. 2016;6:23229. -If a man and a woman are having difficulty conceiving a child and decide to undergo ICSI treatment using their own gametes, which of the following represents the proper developmental pathway of the sperm cell selected to fertilize the egg during this procedure?

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. -Axonal guidance is the process through which axons reach their proper targets in the brain.  Researchers hypothesize that the wild-type PTP gene product mediates proper axonal guidance by inactivating the Abl gene product.  Which experimental observation would best support this hypothesis?

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. -Patients with CAH exhibit hypoglycemia due to numerous endocrine abnormalities.  In healthy individuals, hypoglycemia will trigger an increase in the serum level of:insulin.epinephrine.glucagon.

Several techniques have been developed to treat blood disorders caused by genetic mutations.  Which of the following methods would be most effective in the treatment of a patient homozygous for a deleterious mutation in a gene coding for a subunit of hemoglobin?

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. -The effect of anaerobic exercise on the oxygen saturation curve in healthy patients is shown in Figure 1.  Which of the following is involved in the change in the oxygen dissociation curve?

Following intron splicing in the nucleus, a mature mRNA molecule consists of 500 nucleotides and is translated into a 110 amino acid protein.  Given this, which of the following statements regarding this mRNA molecule would be true?

A drug that blocks the release of adrenocorticotropic hormone from the anterior pituitary would most likely:

A skeletal muscle sarcomere is shown above.  The ability of a resting sarcomere to contract is dependent on the existence of an overlap between actin and myosin filaments and the presence of a gap between the ends of the actin filaments and the M-line.  Given this, which of the following diagrams best represents a resting sarcomere with the potential to generate the greatest amount of contractile force?

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. -Based on Figure 2, which of the following would most likely reestablish the virulence of S. typhimurium transformed with lac⁺?

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. -Transferrin circulating in the blood consists of 679 amino acids, but it is composed of 698 amino acids when it is post-translationally extracted from liver cells, the primary site of transferrin synthesis.  Given this information, researchers would most likely be able to extract and isolate the shorter transferrin peptide from:

A specific recessive X-linked mutation prevents the formation of sweat glands within the skin.  Individuals heterozygous for this mutation exhibit a mosaic phenotype, with some patches of skin lacking sweat glands and other patches containing a normal distribution of sweat glands.  Which of the following statements best explains the genetic mechanism underlying this mosaic phenotype in heterozygous individuals?

Passage Osteoarthritis (OA) is a disorder marked by the deterioration of articular (hyaline) cartilage, the connective tissue lining the ends of bones at most movable joints.  The progressive erosion of articular cartilage in OA is thought to be caused by an inflammatory response induced by years of biomechanical stress due to excessive compression and friction.  This response involves the release of proteolytic enzymes that break down cartilaginous proteins.  The progressive breakdown of articular cartilage ultimately results in complete exposure of the cortical portion of the underlying subchondral bone with narrowing of the joint space.  Repeated and abnormal joint stress can also cause the development of osteophytes, or bony growths, that form on the subchondral bone.  These degenerative processes cause pain and restrict joint movement.Stem cell therapy, in which injected or grafted stem cells are induced to differentiate into chondrocytes (cartilage cells), is a potential new treatment for OA.  To further understand chondrogenesis, researchers have been studying the molecules that regulate the expression and activity of the transcription factor Sox9, which has been shown to promote chondrocyte differentiation.  For example, researchers have found that binding of the growth factor TGF-β to its receptor results in Sox9 activation.MicroRNAs (miRNAs), which are known to alter gene expression by acting at the translational level, are also potential regulators of Sox9.  In particular, miRNA-145 is thought to regulate Sox9 expression by binding to the 3′-UTR of the Sox9 mRNA transcript.  After chondrocyte differentiation was induced using TGF-β, researchers used Western blotting to generate an expression profile of Sox9 and β-actin (a protein not regulated by miRNA-145) in mouse stem cells transfected with miRNA-145, anti-miRNA-145, or neither (Figure 1). Figure 1  Expression of Sox9 in the presence and absence of miRNA-145 or anti-miRNA-145 following induction of chondrocyte differentiation Adapted from Yang B, Guo H, Zhang Y, Chen L, Ying D, Dong S. MicroRNA-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9. PLoS ONE. 2011;6(7):e21679. -Which of the following statements is NOT true about hyaline cartilage?

Passage The nicotinic acetylcholine receptor (nAChR) is a protein embedded in the muscle fiber membrane that facilitates cell-to-cell communication across the neuromuscular junction.  When acetylcholine (ACh) binds nAChR, electrical depolarization within skeletal muscle fibers is induced and ultimately gives rise to contractile activity.  The nAChR is composed of five protein subunits symmetrically organized around a central ion pore.  Previous studies demonstrated that these subunits may serve as epitopes (antibody binding sites) in autoimmune disease.  By reversibly binding nAChR subunits, anti-nAChr antibodies may temporarily block the attachment site for ACh, resulting in muscle weakness.Researchers harvested muscle samples from mice to investigate the effects of anti-nAChR antibodies on muscle tension, the pulling force transmitted by muscles during contraction.Experiment 1First, skeletal muscle samples (tibialis anterior) of adult male mice were surgically harvested (n = 30), washed, and stripped of connective tissues.  Next, the isolated muscle samples were placed in a 150-mL oxygen-infused, electrolyte-rich bath containing glucose and Ca²⁺ ions that were able to enter the muscle fibers and enable maximal contractile force.  One end of the submerged muscle was anchored in place while the other was attached to an apparatus that measures muscle tension and modifies muscle length.  Using electrodes, action potentials were elicited in the harvested muscle tissue to evaluate contractile function (Figure 1). Figure 1  Action potential elicited from a harvested skeletal muscle sampleExperiment 2The researchers divided the 30 muscle samples into three groups of 10.  Resting muscle tension was measured, and the electrolyte-rich baths of each group were further infused with a 7.5-mL solution containing mouse-specific anti-nAChR antibodies, ACh, or a mixture of both (Table 1).  Post-infusion (peak) muscle tension was measured for 30 minutes, with results displayed in Figure 2.Table 1  Solutions Infused into the Electrolyte-Rich Bath Figure 2  Pre-infusion resting muscle tension and post-infusion peak muscle tension in harvested muscle samples (Note: * indicates p < 0.05.) -To further study anti-nAChR antibody function, the researchers wanted to replicate the experiments described in the passage in other mammalian animal models, but their research proposal was rejected by other scientists.  Why was the proposal rejected?