Exam 3: Biology

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

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 -A scientist studying the sequence of the homodimer of LPL would most likely expect that:

Passage Obesity is a condition that affects over 30% of adults in the United States and is associated with comorbidities such as type 2 diabetes mellitus, heart disease, and cancer.  For individuals with clinically severe obesity, defined as having a body mass index above 40 kg/m², lifestyle interventions may produce insufficient weight reduction.  Weight loss surgery, such as Roux-en-Y gastric bypass (RYGB), has proved to be the most effective clinical intervention for facilitating massive and immediate weight loss in clinically obese patients.In RYGB, the stomach is first divided into a small gastric pouch and a distal gastric remnant, which remains attached to the duodenum.  Next, the jejunum is transected and its lower section is connected to the newly created gastric pouch to form the alimentary limb.  This connection is typically created with surgical staples.  The formation of the alimentary limb allows ingested food to bypass the region composed of the distal gastric remnant, the duodenum, and the upper jejunum; collectively termed the biliopancreatic limb.  Finally, the lumen of the upper jejunum is reconnected to the lumen of the lower jejunum to allow enzymes and compounds to flow freely from the biliopancreatic limb into the alimentary limb. Figure 1  RYGB surgically divides the digestive tract into the alimentary limb and the biliopancreatic limb, which then merge into the common limb.RYGB facilitates weight loss by reducing the functional volume of the stomach and altering intestinal anatomy to induce nutrient malabsorption.  However, the procedure is associated with several complications, such as steatorrhea, or fatty stools, due to disruption of lipid digestion.  Animal studies also suggest that RYGB promotes the growth of weight loss-inducing gut bacteria.  Researchers have found that transplanting gut microbiota of obese postoperative RYGB mice into nonoperated, obese mice lacking gut flora may reproduce the therapeutic effects of RYGB in transplant-recipient mice. Adapted from Bächler T, Le roux CW, Bueter M. How do patients' clinical phenotype and the physiological mechanisms of the operations impact the choice of bariatric procedure?. Clin Exp Gastroenterol. 2016;9:181-9. -Nonsteroidal anti-inflammatory drugs (NSAIDs) are weakly acidic compounds that normally pass through the acidic environment of the stomach in a nonionized form until they reach the small intestine, where they become ionized in the less acidic lumen.  Ionized NSAIDs, shown below, are more soluble and therefore dissolve faster. Would NSAID solubility in the gastric pouch of postoperative RYGB patients be higher than normal?

Which of the following nucleic acid complexes would undergo correction by the DNA mismatch repair system?

Passage Obesity is a condition that affects over 30% of adults in the United States and is associated with comorbidities such as type 2 diabetes mellitus, heart disease, and cancer.  For individuals with clinically severe obesity, defined as having a body mass index above 40 kg/m², lifestyle interventions may produce insufficient weight reduction.  Weight loss surgery, such as Roux-en-Y gastric bypass (RYGB), has proved to be the most effective clinical intervention for facilitating massive and immediate weight loss in clinically obese patients.In RYGB, the stomach is first divided into a small gastric pouch and a distal gastric remnant, which remains attached to the duodenum.  Next, the jejunum is transected and its lower section is connected to the newly created gastric pouch to form the alimentary limb.  This connection is typically created with surgical staples.  The formation of the alimentary limb allows ingested food to bypass the region composed of the distal gastric remnant, the duodenum, and the upper jejunum; collectively termed the biliopancreatic limb.  Finally, the lumen of the upper jejunum is reconnected to the lumen of the lower jejunum to allow enzymes and compounds to flow freely from the biliopancreatic limb into the alimentary limb. Figure 1  RYGB surgically divides the digestive tract into the alimentary limb and the biliopancreatic limb, which then merge into the common limb.RYGB facilitates weight loss by reducing the functional volume of the stomach and altering intestinal anatomy to induce nutrient malabsorption.  However, the procedure is associated with several complications, such as steatorrhea, or fatty stools, due to disruption of lipid digestion.  Animal studies also suggest that RYGB promotes the growth of weight loss-inducing gut bacteria.  Researchers have found that transplanting gut microbiota of obese postoperative RYGB mice into nonoperated, obese mice lacking gut flora may reproduce the therapeutic effects of RYGB in transplant-recipient mice. Adapted from Bächler T, Le roux CW, Bueter M. How do patients' clinical phenotype and the physiological mechanisms of the operations impact the choice of bariatric procedure?. Clin Exp Gastroenterol. 2016;9:181-9. -Obese mice underwent either RYGB or sham (placebo) surgery, and two weeks post-surgery, their fecal matter was transplanted into the guts of nonoperated, obese mice lacking gut flora (germ-free recipients).  The body weight of RYGB flora recipients (RYGB-R) and sham surgery flora recipients (SHAM-R) was measured for 3 weeks postcolonization.  Which graphic shows the expected result of this experiment?

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 -A student isolates the cellular components of OPCs in wild-type (WT) mice and in mutant (MT) mice exhibiting a single base substitution that causes reduced osteoclast activity.  Western blot analysis is then performed to test for NF-kB and c-Fos expression in the cytoplasm and nucleus of OPCs isolated from these mice.  The results of this analysis are shown below. Based on the passage, which statement best explains these results?

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

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. -A 10-day-old male was diagnosed with 21-hydroxylase deficiency despite having normal physical examination results.  Which of the following best explains why physical examination results can be normal in males with this condition?

Cirrhosis of the liver is characterized by hepatic cell death, tissue scarring, and altered liver function.  All of the following would be disrupted by cirrhosis of the liver EXCEPT:

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. -The results of Experiment 1 indicate that the oocytes stained blue are:

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. -What conclusion can be made from the results shown in Figure 3?

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. -Given that the fusion of male and female gametes results in fertilization, which statement best explains how unequal crossing over during gametogenesis can lead to 21-hydroxylase deficiency in the fertilized embryo?  One of the parental gametes:

Dehydration caused by frequent loose stools is often observed in patients with certain diseases of the gastrointestinal (GI) tract.  Given this, which of the following treatments would be most effective in treating this symptom?

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 2, ANGPTL4 mutations were found to decrease CAD risk by significantly altering which of the following?LDL cholesterol levelsHDL cholesterol levelsTriglyceride levels

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. -Microscopic examination of tissue samples obtained from the type of bone that becomes exposed in advanced OA is most likely to reveal all of the following EXCEPT:

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) -Progesterone concentration is generally lowest in the proliferative phase and highest in the secretory phase of the menstrual cycle.  The investigators conclude that VEGF concentrations within peritoneal fluid may be inversely correlated with serum levels of progesterone.  Do the data support this conclusion?

Human sperm and oocytes are substantially different in all of the following aspects EXCEPT:

Passage Renal reabsorption of glucose is facilitated by sodium-glucose linked transporters (SGLTs), which are secondary active transport proteins located in the apical membrane of the proximal tubular cells in the kidney.  SGLT activity determines the transport maximum of glucose T_m, the maximum rate at which the kidneys can reabsorb glucose.Excretion of glucose in the urine (glycosuria) occurs when the glomerular filtration rate (GFR) of glucose exceeds the T_m.  Glycosuria is a symptom of diabetes mellitus, a disease marked by hyperglycemia (elevated serum glucose).  Uncontrolled hyperglycemia can damage the nerves that innervate urinary tract musculature, resulting in urinary retention (inability to fully empty the bladder) or incontinence (involuntary urine leakage).Canagliflozin is a SGLT inhibitor used for the treatment for diabetes.  The effect of canagliflozin on renal glucose reabsorption was tested in a glucose infusion experiment in patients with type 2 diabetes mellitus (T2DM).  First, researchers measured the participants' fasting serum glucose levels (average ~80 mg/dL) and then began a continuous intravenous infusion of a glucose solution.  The rate of glucose infusion was adjusted such that blood glucose concentration steadily rose to 300 mg/dL.  Blood and urine glucose concentrations were measured at 30-minute intervals.  The experiment was later repeated in the same patients following canagliflozin administration (100 mg, once a day for seven days).  The results of both trials are shown in Figure 1. Figure 1  Mean urine glucose excretion versus blood glucose concentration in patients with T2DM, with and without canagliflozin treatmentResearchers then estimated the new T_m of glucose following canagliflozin treatment (Figure 2).  Canagliflozin was found to also lower fasting blood glucose in individuals with T2DM.  Side effects included weight loss, increased urinary frequency, excessive thirst, and blood pressure reduction. Figure 2  Mean rate of renal glucose reabsorption versus plasma glucose concentration in patients with T2DM, with and without canagliflozin treatment -Researchers hypothesized that administration of canagliflozin reduces blood pressure in patients with diabetes by increasing urine output and reducing total blood volume.  A decrease in which of the following would also result in lowered blood pressure?Activation of the renin-angiotensin systemProduction of aldosteroneSecretion of antidiuretic hormone

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 conclusions regarding virulence of lac⁺ S. typhimurium is most accurate?

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. -If changes in p21 expression contribute to the development of B-cell lymphoma in Eμ-Myc mice, what is the expected result of real-time PCR analysis of c-Myc and p21 expression in wild-type (wt) and Eμ-Myc (Eμ) mice?  (Note: mRNA levels are standardized to the expression of ubiquitin (Ub), which is not regulated by c-Myc.)