Exam 3: Biology

Passage The sex chromosomes X and Y are involved in mammalian sex determination.  Organisms with two wild-type (WT) X chromosomes develop as females (XX), but those with a combination of WT X and Y chromosomes develop as males (XY) (Table 1).  Both sexes also inherit a set of autosomes (nonsex chromosomes).  Humans inherit 22 pairs of autosomes (44 total).  In contrast, rabbits inherit 21 pairs of autosomes (42 total).Table 1  Chromosome Types Inherited by a Male and Female Rabbit (Note: The Y chromosome has fewer alleles than the X chromosome.) Specifically, the SRY gene, located on the mammalian Y chromosome, is crucial for male sex determination and induction of testis development.  Studies in mammals have shown that regardless of an organism's combination of sex chromosomes, fetuses will always develop as female in the absence of the SRY protein.  In addition, one of the X chromosomes becomes inactivated in WT,XX females to prevent them from having double the amount of X-chromosome gene products as WT,XY males.Sp1 is a transcription factor with demonstrated regulatory control of SRY gene expression, and XY individuals with mutations affecting the Sp1 binding site on the SRY gene have been shown to develop female sex characteristics.  Unlike SRY, the gene for Sp1 is located on an autosome.Researchers knocked out (KO) Sp1 binding sites on the SRY gene in rabbit embryos (Sp1 KO,XY rabbits) and observed their sexual development following implantation in surrogate mothers.  The relationship between the SRY gene and sexual development can be explained by the following experimental observations: Most Sp1 KO,XY rabbits had female genitalia and displayed normal mating behaviors. Sp1 KO,XY rabbits had significantly smaller ovaries and fewer follicles than WT,XX rabbits. No significant differences were observed in the cervix and uterus between Sp1 KO,XY and WT,XX rabbits. There was significantly reduced SRY expression in Sp1 KO,XY rabbits when compared to WT,XY males. WT,XY male rabbits had significantly higher testosterone levels when compared to Sp1 KO,XY rabbits. Sp1 KO,XY rabbits were infertile when mated with WT,XY males. Sp1 KO,XY rabbits could become pregnant via embryo transfer. Song Y et al. Mutation of the Sp1 binding site in the 5' flanking region of SRY causes sex reversal in rabbits. Oncotarget. 2017; 8(24):38176-83. -Based on the information given in the passage, which of the following observations best supports the conclusion that the second X chromosome that WT,XX females inherit affects their fertility?

In neurons, action potential propagation occurs along the axon.  The analogous structure in skeletal muscle fibers that enables action potential propagation is the:

Passage Abnormal blood lipid levels play a significant role in the development of coronary artery disease (CAD).  The discovery of genomic variations that confer a protective effect against CAD has prompted investigations into the biological impact of these protective alleles.  Researchers have observed that specific single-base variations in the ANGPTL4 gene are common among patients with a low occurrence of CAD.  ANGPTL4 is located on chromosome 19 and codes for angiopoietin-like 4, a multifunctional glycosylated protein.One of the functions of angiopoietin-like 4 is the regulation of lipid metabolism.  The N-terminal domain of angiopoietin-like 4 induces a conformational change in the enzyme lipoprotein lipase (LPL), forcing the LPL homodimer to dissociate into two inactive 225-residue monomers.  LPL functions on the surface of vascular endothelial cells and degrades the triglycerides of chylomicrons and very-low-density lipoproteins (VLDLs) in the blood.  Elevated triglyceride levels have been correlated with increased CAD risk.To identify common loss-of-function mutations in ANGPTL4, DNA samples from 43,000 individuals with CAD and 78,000 individuals without CAD were genotyped.  A detailed analysis of ANGPTL4 genomic variants was performed, and the results of individuals without CAD were mapped in Figure 1.  The lipid profiles of ANGPTL4 mutation noncarriers were also compared to those of mutation carriers (Figure 2). Figure 1  Loss-of-function mutations observed in ANGPTL4 in participants without CAD  (Note:  * = mutation resulting in a truncated protein; † = substitution of a cytosine for its complementary base.) Figure 2  Mean plasma lipid levels for ANGPTL4 mutation carriers and noncarriers (Note:  LDL = low-density lipoprotein; HDL = high-density lipoprotein; error bars = standard deviation.)To further analyze the effect of ANGPTL4 on blood lipid levels, ANGPTL4 knockout mice and mice that constitutively express ANGPTL4 were created.  Researchers hypothesized that two of the four biological profiles shown in Table 1 would be observed in these mice.Table 1  Predicted Biological Profiles of ANGPTL4 Mouse Models -Based on Figure 1, which of the following nucleotides would cause the T291-1 loss-of-function mutation if it were substituted for cytosine?

Passage The heart is a muscular organ comprising four chambers, two atria and two ventricles, that function synergistically to pump blood through a vast, closed network of blood vessels.  The chambers are separated by membranous muscular barriers known as septa.  Oxygenated blood returning from the lungs via the pulmonary veins fills the left atrium and enters the left ventricle, which then pumps the blood into the systemic arteries to supply other organs in the body.  By contrast, deoxygenated blood returning from systemic veins first enters the right atrium, then the right ventricle, and is ultimately siphoned into the pulmonary arteries leading to the lungs.  The necessary oxygen, nutrient, and waste exchange occurs between the thinnest blood vessels (capillaries) and neighboring tissues of the systemic and pulmonary circuits.Blood flow throughout the circulatory system is dictated by blood (hydrostatic) pressure, vascular resistance (force opposing blood flow through a vessel), and cardiac output (blood volume expelled from the ventricles per unit time).  When blood traverses a vessel, it exerts hydrostatic pressure on the vessel walls, which results in the forced movement of fluid out of the vessel and into the interstitial space.  The circulating plasma proteins cause the osmotic pressure within the vessel to be higher than that of the interstitial fluid.  In turn, osmotic pressure causes fluid to flow from the interstitial space into the blood vessel, opposing hydrostatic pressure.Cardiac output depends in part on heart rate, which is tightly regulated by the sinoatrial (SA) and atrioventricular (AV) nodes, specialized groups of self-depolarizing cells located in the upper right atrial wall and lower interatrial septum, respectively.  Action potentials (APs) generated by SA nodal cells stimulate atrial contraction as they travel to the AV node.  The AV node delays AP transmission to ventricular cells, ensuring ventricular filling is complete prior to heart contraction.A 63-year-old male patient collapsed while exercising and was hospitalized.  The patient presented with an elevated heart rate, low systemic blood pressure, and abnormally low blood oxygen levels.  In addition, x-rays revealed excess fluid in his lungs. -The attending physician orders an electrocardiogram (ECG) to measure the electrical activity of the patient's heart.  The results of the ECG of a healthy individual at rest (A) and the ECG of the patient at rest (B) are shown below. Which of the following conclusions regarding the patient's cardiac function is best supported by these results?

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. -Which of the following genes would be the most advantageous for lac⁺ S. typhimurium bacteria in glucose-rich media?

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. -Recently fed ob/ob mice show deficient binding of glucagon to its G protein-coupled receptor, which most likely leads to:

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 -EHEC is classified as a bacilli bacterium.  Given this information, this type of classification is most likely based on the bacterium's:

A scientist claims that endurance training is an exercise method that can improve physical performance by inducing changes in type 1 skeletal muscle fibers that allow the fiber to perform aerobic respiration more efficiently to generate ATP.  Given this, which of the following adaptations is most likely to occur in type 1 fibers in response to endurance training?Mitochondrial density of the muscle fibers increasesCapillary supply around the muscle fiber increasesMyoglobin concentration per muscle fiber increases

Impaired release of which of the following neurotransmitters would directly result in the paralysis of skeletal muscles?

Following digestion in the small intestine, certain lipids are absorbed by intestinal epithelial cells and packaged into large droplets.  These lipid droplets are released from intestinal epithelial cells and transported into the lymphatic system to ultimately drain into a large vein near the heart.  Which series shows the order in which these lipid droplets are transported from the intestine to the bloodstream?

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. -Undifferentiated gastrula cells presumed to give rise to neural tissue were transplanted from a donor frog embryo onto an undifferentiated gastrula area of a host frog embryo.  The transplantation region in the host was presumed to give rise to epidermal tissue.  Both host and donor embryos were allowed to develop following the transplant, and gave rise to two normal embryos.  What was the purpose of this experiment?

Passage Viruses are known as obligate intracellular parasites because they cannot reproduce independently (outside of a host cell).  Viral genomes (RNA or DNA) are enclosed by a protein shell known as the capsid.  Some viruses, known as enveloped viruses, have an additional phospholipid bilayer that fully envelops the capsid.  Viruses that lack this outermost phospholipid covering are labeled non-enveloped.  The entry of either enveloped or nonenveloped viruses into a host cell enables the production of viral proteins and the subsequent assembly of viral progeny.Immune cells (B and T cells) protect host organisms against viral infections by recognizing viral particles (antigens) as foreign.  In response to the recognition of foreign particles, antigen-presenting cells display cleaved antigens (also known as antigen fragments) on their surface, which enable the activation of other immune cells that stimulate an immune response, including the production of neutralizing antibodies.An example of an infectious disease is viral hepatitis (inflammation of the liver), which can be caused by any one of five distinct hepatitis viruses (A-E) that infect hepatocytes (liver cells).  HAV, HCV, and HEV contain positive-sense, single-stranded RNA (+ssRNA) as genetic material.  In contrast, HBV has a partially double-stranded circular DNA genome.  HDV is unique because it has a negative-sense, single-stranded RNA (−ssRNA) genome.Researchers hypothesized that synthetic antibodies could be used to treat viral hepatitis in patients with compromised immune systems.  Fluorescent antibodies, designed to bind the capsid proteins of the five hepatitis viruses, were added to separate wells containing each type of virus.  The fluorescence assay that was performed detected only bound antibodies in media with HAV or HEV. Adapted from Shin EC, Sung PS, Park SH. Immune responses and immunopathology in acute and chronic viral hepatitis. Nat Rev Immunol. 2016;16(8):509-23. -An individual with hepatitis is suspected to have been exposed to either a bacterial strain of Bartonella henselae (0.5 µm) or HCV (0.055 µm).  Healthy hepatocytes and hepatocytes infected with serum from the affected patient were grown on chamber slides separated by double membranes, as shown below. A cellular morphology similar to infected hepatocytes was observed in both chambers 2 and 3 after a 6 hour incubation time.  Using a light microscope, can investigators observe the agent that caused the patient's hepatitis in chamber 3?

Which statement most accurately describes the role of T tubules in skeletal muscle cells?

Passage Saliva, a secreted fluid with multiple physiologic functions, is composed of water (~99%), proteins, and electrolytes.  Together, these components facilitate nutrient consumption, respiration, and communication.  For example, saliva aids in digestion by serving as a lubricant during mastication and by initiating carbohydrate and fat metabolism.  In addition, the calcium and phosphate present in saliva promote tooth remineralization, and antimicrobial and chemical buffering agents give saliva protective properties.The gustatory-salivary reflex, the secretion of saliva from the salivary glands in response to gustation (taste), is primarily mediated by the parasympathetic division of the nervous system.  In general, parasympathetic pathways originate from the brain stem or the sacral spinal cord.  Fibers originating from nuclei in these regions synapse onto ganglia that lie in close proximity to the target organ.  Both preganglionic neurons (those originating from the nuclei) and postganglionic neurons (those originating from the ganglia) release acetylcholine (ACh).In the gustatory-salivary reflex, activation of taste receptors in the tongue sends impulses to the superior and inferior salivatory nuclei in the brainstem.  Local processing within these nuclei activates the parasympathetic pathways that innervate the salivary glands.  Ultimately, ACh released from postganglionic neurons acts on the muscarinic acetylcholine receptors (mAChRs) on the secretory cells of the salivary gland to stimulate salivation (Figure 1). Figure 1  Parasympathetic regulation of salivary secretionSympathetic stimulation of the salivary glands also promotes salivation but to a lesser degree than parasympathetic stimulation.  This may contribute to what is often perceived to be decreased salivation when the sympathetic division is more active than its counterpart.  Unlike parasympathetic pathways, sympathetic pathways originate in the thoracic and lumbar spinal segments, and the ganglia lie in close proximity to the spinal cord.  Although preganglionic neurons of the sympathetic nervous system release ACh, the postganglionic neurons often release norepinephrine. -The graph below shows the firing rate of preganglionic neurons within the rat gustatory-salivary reflex arc when the lateral hypothalamic area (LHA) is electrically stimulated at various time intervals before the tongue is stimulated.  The results are compared to the preganglionic firing rate following tongue-only stimulation, which was used as a baseline that represented 100% activity. The results of this experiment indicate that at shorter time intervals:

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. -Primary ciliary dyskinesia (PCD), a rare disease that impairs the normal function of cilia, is often misdiagnosed as atypical asthma because wheezing is observed in both illnesses.  Unlike asthma, PCD would cause the inability to:

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. -What steps are required to produce a mature FL-SMN transcript?Excision of noncoding sequences between the 3′ and 5′ untranslated regions in pre-mRNAAddition of multiple adenine nucleotides to the 3′ end of SMN1 pre-mRNARemoval of the 7-methylguanosine cap from mature mRNADNA polymerase binding to the SMN1 gene

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 -Which of the following sequences accurately describes the pathway of communication between neurons?

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. - The diagram above represents the immature gamete of a male fly that is heterozygous for Ena.  If the gamete undergoes meiotic division and Ena alleles do not undergo recombination, during which phase of meiosis will the wild-type Ena alleles be pulled to opposite poles of the cell?

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 -Based on the passage and the experimental results shown in Figure 1, which event is an outcome of treating hCaSR cells with AMG 416?

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. -OA commonly affects large, weight-bearing joints of the lower extremities such as the knee and hip.  Based on the passage, which of the following would occur in a patient with OA involving the knees?