Serial Ultrasonography for the Assessment of Healing of Lower Extremity Bone Stress Injury and Correlation With Return to Sport/Exercise.

BACKGROUND: Lower extremity bone stress injuries (BSIs) are common among athletes who participate in high-impact activities. Conventional imaging is limited in assessing healing of BSIs. HYPOTHESIS: Serial ultrasonography (US) can identify changes in appearance of lower extremity BSIs over time that can be correlated with symptoms and return to exercise/sport. STUDY DESIGN: Cohort observational study. LEVEL OF EVIDENCE: Level 3. METHODS: Adults 18 to 50 years old with a recent exercise-associated BSI of distal tibia/fibula or metatarsals diagnosed by magnetic resonance imaging (MRI) were enrolled. US was performed every 2 weeks for 12 weeks. The sonographic appearance (soft tissue edema, periosteal reaction, hyperemia on power Doppler, callus) was correlated with the numerical rating scale (NRS) for pain and ability to return to sport/exercise. RESULTS: A total of 30 patients were enrolled (mean age, 35.3 ± 7.7 years; 21 [70.0%] female). The tibia was most frequently affected (n = 15, 50.0%), followed by metatarsals (n = 14, 46.7%) and fibula (n = 1, 3.3%). At week 4, 25 of 30 (83.3%) had at least 1 US finding associated with the BSI. The degree of hyperemia was correlated with NRS at weeks 4 and 6 (Spearman correlations [ρ] 0.45 [0.09, 0.69] and 0.42 [0.07, 0.67], respectively), as well as return to sport/exercise at week 6 (ρ -0.45 [-0.68, -0.09]). US soft tissue edema was also correlated with NRS at week 6 (ρ 0.38 [0.02, 0.65]). CONCLUSION: Serial US of lower extremity BSIs can provide objective measures of healing. US findings were correlated with clinical outcomes at multiple timepoints. CLINICAL RELEVANCE: US may have advantages over conventional imaging for monitoring healing of lower extremity BSIs. Further research is needed to better understand the prognostic value of these sonographic indicators of BSI healing and role in assessing readiness for return to sport/exercise.

Child Sexual Abuse of Elite Athletes: Prevalence, Perceptions, and Mental Health.

Despite a series of high-profile media reports of sexual abuse in sport over the past few years, little research has been done to explore the scope of the problem in the United States. The current article reports on prevalence of child sexual assault in elite athletes in the United States. Using a retrospective web survey, adults answered questions on their experiences in sport. Of the 473 elite athletes surveyed, 3.8% (n = 18) reported being sexual assaulted as a minor in the sporting context. Of those reporting assault, most (61%) reported being abused by an adult authority figure (usually a coach) and 44% reported being assaulted by a peer. Abused athletes were significantly more likely to report having been diagnosed with a mental disorder (Fisher's exact test; p < .001). The findings can be utilized to improve prevention and child protection measures and other safeguarding initiatives in sport.

Analysis of the Written Policies of the 32 U.S. Archdioceses on the Prevention of Child Sexual Abuse.

The Catholic Church in the U.S. has been under concerted public pressure to improve its child protection policies. However, development of these policies has largely been left to the discretion of local dioceses with little central oversight. To determine the scope of current abuse prevention policies, we analyzed all 32 U.S. archdioceses' policies and practices for developing a safe environment and preventing child sexual abuse. Using a tool developed from a list of unique components selected from the policies of all 32 archdioceses, we compared policies and procedures across archdioceses. On a group level, we found policies to be inadequate. The average score for archdioceses in the general area of prevention was 40.7 (SD = 9.3) out of 102 possible points (40% of possible points) with few prevention policies conforming to basic best practices that would be expected of a youth-serving organization. The variation and inconsistencies across archdioceses reveal the need for the Catholic Church to establish more uniform standards for preventing sexual abuse. These standards should be grounded in evidence-based practices and expert guidance. We also discuss some key areas which warrant immediate attention in future policy-making.

Prevalence of Vitamin D Insufficiency and Deficiency in Young, Female Patients With Lower Extremity Musculoskeletal Complaints.

BACKGROUND: Low levels of vitamin D have well-known impacts on bone health, but vitamin D also has a more global role throughout many tissues, including skeletal muscle. The high prevalence of hypovitaminosis D and the vast physiological features of vitamin D have led researchers to examine the influence of vitamin D on physical performance and injury. Because of the critical role of vitamin D in maintaining musculoskeletal health and function, a high rate of hypovitaminosis D among female patients with a variety of musculoskeletal issues could be of high clinical relevance. HYPOTHESIS: There is a high prevalence of low vitamin D in female patients with both acute and overuse sports-related issues of both soft tissue and bone. STUDY DESIGN: Prospective cohort study. LEVEL OF EVIDENCE: Level 3. METHODS: Female patients, aged 16 to 40 years, presenting with lower extremity injury diagnosed within the past 4 weeks, no use of multivitamin or vitamin D supplement, and no history of malabsorption syndrome met the inclusion criteria. Vitamin D levels were assessed and categorized as normal (≥32 ng/mL) and low, which includes insufficient (20.01-31.9 ng/mL) and deficient (≤20 ng/mL). RESULTS: Of the 105 patients enrolled, 65.7% had low vitamin D. Within the low vitamin D cohort, 40.6% were deficient and 59.4% were insufficient. Injuries were grouped into overuse or acute with 74 overuse injuries and 31 acute injuries, exhibiting low vitamin D prevalence of 60.8% and 77.4%, respectively. Patients with ligamentous/cartilaginous injuries exhibited the highest percentage of low vitamin D (76.5%), followed by those with patellofemoral-related complaints (71.0%), muscle/tendon injuries (54.6%), and bone stress injuries (45.5%). In univariable analysis, older age, non-White race, less physical activity, less high-intensity interval training days, less endurance training days, and more rest days showed an association with low vitamin D, but none showed an independent association in multivariable analysis. CONCLUSION: The prevalence of low vitamin D in female patients with various musculoskeletal complaints was high. Clinicians should evaluate for low vitamin D in both acute and overuse injuries. CLINICAL RELEVANCE: In addition to screening, this study suggests that clinicians should evaluate for low vitamin D levels beyond bone stress injuries in the setting of acute and overuse injuries.

Menstrual Irregularity, Hormonal Contraceptive Use, and Bone Stress Injuries in Collegiate Female Athletes in the United States.

BACKGROUND: Menstrual irregularity (MI) is common in female athletes and is a component of the Female Athlete Triad (Triad). Many athletes with the Triad are started on hormonal contraceptives (HC) for MI, but this interferes with the ability to monitor menstrual cycle regularity and can mask other causes of MI. There are limited studies investigating the relationship between MI, HC use, and injury in female collegiate athletes. OBJECTIVE: To examine the prevalence of and relationship between HC use, MI, and bone stress injuries in female collegiate athletes in the United States. DESIGN: Cross-sectional study. SETTING: Online survey. PARTICIPANTS: 1020 U.S. female collegiate athletes (age ≥ 18 years). METHODS OR INTERVENTIONS: Assessment of risk factors for menstrual irregularity and bone stress injuries was conducted via a one-time survey. MAIN OUTCOME MEASURES: HC use, MI, history of stress fractures. RESULTS: Current HC use prevalence was 65% (95% confidence interval [CI], 61.9%, 67.8%). Of all athletes, 47% reported past MI. Of the athletes who were not currently using HCs, 32% had current MI. Compared with athletes without past MI, more athletes with past MI reported current HC use (73% vs. 57%) and indicated menstrual cycle consistency as the primary reason for use (24% vs. 4%) (P < .001). Additionally, 25% of athletes reported a history of stress fractures, which was associated with lean/aesthetic sports participation (odds ratio [95% CI]: 1.9 [1.4, 2.5]; P < .001) and less oral contraceptive pill (OCP) use (0.7 [0.5, 1.0]; P = .043). Compared with OCPs, injectable HCs were associated with greater odds of a history of stress fractures (4.5[1.6, 12.3]; P = .004). CONCLUSIONS: HC use was prevalent among this cohort of female collegiate athletes, and almost half of the athletes reported past MI. A goal of menstrual cycle regularity was cited by 24% of athletes as a primary reason to use HCs, which shows that more athlete education is needed to avoid masking MI and the Triad with HCs. Further studies elucidating the relationship between HC use, MI, and sports-related injury are warranted.

High Number of Daily Steps Recorded by Runners Recovering from Bone Stress Injuries.

BACKGROUND: Bone stress injuries (BSIs) are common among runners for which activity modification is the primary treatment. The clinical utility of measuring activity during recovery has not been evaluated. QUESTIONS/PURPOSES: We sought to measure the physical activity of runners recovering from BSIs and determine if activity can be correlated with symptoms. METHODS: A prospective observational pilot study was performed of runners with a new lower extremity BSI treated non-surgically. For 30 days, activity of runners was measured with a physical activity tracker and daily pain scores were collected. RESULTS: We enrolled 18 runners (average age, 33 years; 72% female). Twelve had stress fractures and six had stress reactions. The average daily steps of all runners during the observation period was 10,018 ± 3232, and the runner with the highest daily steps averaged 15,976. There were similar average daily steps in those with stress fractures versus reactions, 10,329 versus 9965, respectively. There was no correlation between daily steps or relative change in daily steps with pain or relative change in pain scores. CONCLUSION: Runners with BSIs averaged over 10,000 steps per day during early recovery. Clinicians may not be aware of the amount of activity runners maintain after being diagnosed with a BSI. Although daily steps and symptoms could not be correlated in this study, objectively measuring activity may assist clinicians in guiding runners recovering from BSIs.

Case report: ischial stress fracture non-union in a college football player.

Stress fractures are common injuries associated with repetitive high-impact activities, often in high-level athletes and military recruits. Although predominantly occurring in the lower extremities, stress fractures may occur wherever there is a sudden increase in frequency or intensity of activity, thereby overloading the yield point of the local bone environment. Ischial stress fractures are a rarely diagnosed cause of pain around the hip and pelvis. Often, patients present with buttock pain with activity, which can be misdiagnosed as proximal hamstring tendonitis or avulsion. Here, we report a case of a college football player who was diagnosed with an ischial stress fracture which went on to symptomatic non-union after extensive conservative management. We treated his ischial non-union with open reduction internal fixation utilizing a tension band plate and screws. This interesting case highlights an uncommon cause of the relatively common presentation of posterior hip pain and describes our technique for addressing a stress fracture non-union in the ischium.

The barriers to a national inquiry into child sexual abuse in the United States.

There is an often-overlooked but critical factor at the center of institutional child sexual abuse that must be acknowledged and addressed: adults tend to place the interest of institutions and other adults above the protection of children. As the Australian Royal Commission into Institutional Responses to Child Sexual Abuse has shown, this phenomenon is evident across institutional settings and any institutional reform aimed at improving child safety must therefore guard against this tendency if it is to be effective in protecting children. In the United States there are also other barriers to dealing with child sexual abuse in institutional contexts. State government responses to the challenges of child sexual abuse have varied. However, the federal governmsent has been silent on the problem of religious institutional sexual abuse. This commentary considers how the politics of religious liberty in the United States inhibits action by protecting institutions that cover up child sexual abuse.

Transgender Individuals and Osteoporosis Prevention.

BACKGROUND: Risk behaviors and hormone use place transgender individuals (TIs) in jeopardy for osteoporosis. PURPOSE: The purpose of this study was to expand the science about the knowledge, health beliefs, and osteoporosis (OP) preventing behaviors of TIs. METHODS: This was a mixed-methods pilot study of a convenience sample of 31 TIs 30 years and older. Participants completed an online Osteoporosis Bone Health Survey. Fifteen participants were randomly selected for an interview to describe their perceptions of bone health. RESULTS: Transgender individuals performed poorly on the knowledge measure. There were no significant findings for osteoporosis health beliefs. Participants' daily dietary dairy calcium intake was 800 mg and daily walking activity was 17 minutes. Perceptions of bone health revealed two essential elements, knowing and doing. CONCLUSION: Determining TIs' bone health perception is important because of their unique healthcare issues. Healthcare providers need to be aware of TIs' bone health needs to help enhance TIs' OP knowledge, health beliefs, and preventing behaviors.

Bone Health in Athletes.

CONTEXT: The health of the skeletal system is important for athletes young and old. From the early benefits of exercise on bones to the importance of osteoporosis prevention and treatment, bone health affects the ability to be active throughout life. EVIDENCE ACQUISITION: PubMed articles dating from 1986 to 2016 were used for the review. Relevant terms such as keywords and section titles of the article were searched and articles identified were reviewed for relevance to this article. STUDY DESIGN: Clinical review. LEVEL OF EVIDENCE: Levels 1 through 4 evidence included. RESULTS: There is strong evidence that exercise benefits bone health at every age and is a critical factor in osteoporosis prevention and treatment. Vitamin D, calcium, and hormones play vital roles in ensuring optimal bone health. When there is an imbalance between exercise and nutrition, as seen in the female athlete triad, bone health is compromised and can lead to bone stress injuries and early osteoporosis. Both of these can lead to morbidity and lost time from training and competition. Thus, early recognition and appropriate treatment of the female athlete triad and other stress fracture risk factors are vital to preventing long-term bone health problems. CONCLUSION: To optimize bone health, adequate nutrition, appropriate weightbearing exercise, strength training, and adequate calcium and vitamin D are necessary throughout life.

Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells.

Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin-mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.

Correlation of MRI grading of bone stress injuries with clinical risk factors and return to play: a 5-year prospective study in collegiate track and field athletes.

BACKGROUND: Bone stress injuries are common in track and field athletes. Knowledge of risk factors and correlation of these to magnetic resonance imaging (MRI) grading could be helpful in determining recovery time. PURPOSE: To examine the relationships between MRI grading of bone stress injuries with clinical risk factors and time to return to sport in collegiate track and field athletes. STUDY DESIGN: Cohort study (prognosis); Level of evidence, 2. METHODS: A total of 211 male and female collegiate track and field and cross-country athletes were followed prospectively through their competitive seasons. All athletes had preparticipation history, physical examination, and anthropometric measurements obtained annually. An additional questionnaire was completed regarding nutritional behaviors, menstrual patterns, and prior injuries, as well as a 3-day diet record. Dual-energy X-ray absorptiometry was performed at baseline and each year of participation in the study. Athletes with clinical evidence of bone stress injuries had plain radiographs. If radiograph findings were negative, MRI was performed. Bone stress injuries were evaluated by 2 independent radiologists utilizing an MRI grading system. The MRI grading and risk factors were evaluated to identify predictors of time to return to sport. RESULTS: Thirty-four of the athletes (12 men, 22 women) sustained 61 bone stress injuries during the 5-year study period. The mean prospective assessment for participants was 2.7 years. In the multiple regression model, MRI grade and total-body bone mineral density (BMD) emerged as significant and independent predictors of time to return to sport. Specifically, the higher the MRI grade (P = .004) and lower the BMD (P = .030), the longer the recovery time. Location of the bone injury at predominantly trabecular sites of the femoral neck, pubic bone, and sacrum was also associated with a prolonged time to return to sport. Female athletes with oligomenorrhea and amenorrhea had bone stress injuries of higher MRI grades compared with eumenorrheic athletes (P = .009). CONCLUSION: Higher MRI grade, lower BMD, and skeletal sites of predominant trabecular bone structures were associated with a delayed recovery of bone stress injuries in track and field athletes. Knowledge of these risk factors, as well as nutritional and menstrual factors, can be clinically useful in determining injury severity and time to return to sport.

The Role of Vitamin D Supplements in Women's Health.

Vitamin D is pivotal to the absorption of calcium and maximizing bone health. Women suffer great morbidity and mortality related to osteoporosis and fractures, which may be decreased by interventions such as vitamin D. In addition, extraskeletal benefits of vitamin D have been postulated including positive effects on cancer. Both the classical and nonclassical functions of vitamin D will be discussed here, with a focus on women.

A displaced femoral neck stress fracture in an amenorrheic adolescent female runner.

This case demonstrates the potential serious consequences of the female athlete triad and its effects on bone. Displaced femoral neck stress fractures cause significant morbidity, and this case highlights the preventable nature of this injury. The treatment was focused on improving low energy availability, and, although challenging, improvements were made. This injury could have been prevented if the signs and symptoms of her injury had been addressed and there had been better knowledge of her risk factors. This case highlights the need for further education in the sports and health communities.

Recent advances in Chlamydia subversion of host cytoskeletal and membrane trafficking pathways.

Chlamydia species are obligate intracellular bacteria that cause sexually transmitted disease, ocular infections and atypical pneumonia. This review highlights recent advances describing the mechanisms by which Chlamydia subvert host cytoskeleton and membrane trafficking pathways to create a replication competent niche.

Multiple host proteins that function in phosphatidylinositol-4-phosphate metabolism are recruited to the chlamydial inclusion.

Chlamydiae replicate within a nonacidified vacuole, termed an inclusion. As obligate intracellular bacteria, chlamydiae actively modify their vacuole to exploit host signaling and trafficking pathways. Recently, we demonstrated that several Rab GTPases are actively targeted to the inclusion. To define the biological roles of inclusion localized Rab GTPases, we have begun to identify inclusion-localized Rab effectors. Here we demonstrate that oculocerebrorenal syndrome of Lowe protein 1 (OCRL1), a Golgi complex-localized phosphatidylinositol (PI)-5-phosphatase that binds to multiple Rab GTPases, localizes to chlamydial inclusions. By examining the intracellular localization of green fluorescent protein (GFP) fusion proteins that bind to unique phosphoinositide species, we also demonstrate that phosphatidylinositol-4-phosphate (PI4P), the product of OCRL1, is present at the inclusion membrane. Furthermore, two additional host proteins, Arf1, which together with PI4P mediates the recruitment of PI4P-binding proteins to the Golgi complex, and PI4KII alpha, a major producer of Golgi complex-localized PI4P, also localize to chlamydial inclusions. Depletion of OCRL1, Arf1, or PI4KII alpha by small interfering RNA (siRNA) decreases inclusion formation and the production of infectious progeny. Infectivity is further decreased in cells simultaneously depleted for all three host proteins, suggesting partially overlapping functions in infected cells. Collectively, these data demonstrate that Chlamydia species create a unique replication-competent vacuolar environment by modulating both the Rab GTPase and the PI composition of the chlamydial inclusion.

The Anaplasma phagocytophilum-occupied vacuole selectively recruits Rab-GTPases that are predominantly associated with recycling endosomes.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell-derived vacuole. The A. phagocytophilum-occupied vacuole (ApV) fails to mature along the endocytic pathway and is non-fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)-tagged Rab GTPases in A. phagocytophilum-infected HL-60 cells. GFP-Rab4A, GFP-Rab10, GFP-Rab11A, GFP-Rab14, RFP-Rab22A and GFP-Rab35, which regulate endocytic recycling, and GFP-Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome-associated GFP-Rabs and acquisition of GFP-Rab5, GFP-Rab7, and the lysosomal marker, LAMP-1. Wild-type and GTPase- deficient versions, but not GDP-restricted versions of GFP-Rab1, GFP-Rab4A and GFP-Rab11A, localize to the ApV. Strikingly, GFP-Rab10 recruitment to the ApV is guanine nucleotide-independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.

Manipulation of rab GTPase function by intracellular bacterial pathogens.

Intracellular bacterial pathogens have evolved highly specialized mechanisms to enter and survive within their eukaryotic hosts. In order to do this, bacterial pathogens need to avoid host cell degradation and obtain nutrients and biosynthetic precursors, as well as evade detection by the host immune system. To create an intracellular niche that is favorable for replication, some intracellular pathogens inhibit the maturation of the phagosome or exit the endocytic pathway by modifying the identity of their phagosome through the exploitation of host cell trafficking pathways. In eukaryotic cells, organelle identity is determined, in part, by the composition of active Rab GTPases on the membranes of each organelle. This review describes our current understanding of how selected bacterial pathogens regulate host trafficking pathways by the selective inclusion or retention of Rab GTPases on membranes of the vacuoles that they occupy in host cells during infection.

Chlamydia pneumoniae inclusion membrane protein Cpn0585 interacts with multiple Rab GTPases.

Chlamydiae are intracellular bacteria that develop within a membrane-bound vacuole called an inclusion. To ensure that the inclusion is a safe niche for chlamydial replication, chlamydiae exploit a number of host cell processes, including membrane-trafficking pathways. Recently, several Rab GTPases were found to associate with the inclusions of various chlamydial species. Here we report that Cpn0585, a Chlamydia pneumoniae inclusion membrane protein (Inc), interacts with multiple Rab GTPases. The results from yeast two-hybrid experiments revealed that an amino-terminally truncated form of Cpn0585 (Cpn0585(102-651)) interacts with Rab1, Rab10, and Rab11 but not with Rab4 or Rab6. Cpn0585-Rab GTPase interactions are direct and GTP dependent as shown in glutathione S-transferase pull-down assays using native and recombinant Cpn0585. In C. pneumoniae-infected HEp-2 cells transfected with enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, the colocalization with Cpn0585 at the inclusion membrane was partial for EGFP-Rab1 and EGFP-Rab10, but extensive for wild-type EGFP-Rab11A and the constitutively active GTPase-deficient EGFP-Rab11AQ70L. Moreover, Cpn0585 colocalized with EGFP-Rab11AQ70L as early as 2 h postinfection. Upon delivery into live C. pneumoniae-infected cells, Cpn0585(628-651)-specific antibodies bound to the inclusion membrane, demonstrating that the Rab GTPase-interacting domain of Cpn0585 faces the host cell cytosol. Finally, ectopic expression of Cpn0585(102-651) partially inhibited the development of C. pneumoniae inclusions in EGFP. but not in EGFP-Rab11AQ70L-expressing HEp-2 cells. Collectively, these data suggest that Cpn0585 is involved in the recruitment of Rab GTPases to the inclusion membrane and that interfering with this function may adversely impact the fitness of the C. pneumoniae inclusion for chlamydial replication.

Divergent functions of VTI12 and VTI11 in trafficking to storage and lytic vacuoles in Arabidopsis.

The protein storage vacuole (PSV) is a plant-specific organelle that accumulates reserve proteins, one of the main agricultural products obtained from crops. Despite the importance of this process, the cellular machinery required for transport and accumulation of storage proteins remains largely unknown. Interfering with transport to PSVs has been shown to result in secretion of cargo. Therefore, secretion of a suitable marker could be used as an assay to identify mutants in this pathway. CLV3, a negative regulator of shoot stem cell proliferation, is an extracellular ligand that is rendered inactive when targeted to vacuoles. We devised an assay where trafficking mutants secrete engineered vacuolar CLV3 and show reduced meristems, a phenotype easily detected by visual inspection of plants. We tested this scheme in plants expressing VAC2, a fusion of CLV3 to the vacuolar sorting signal from the storage protein barley lectin. In this way, we determined that trafficking of VAC2 requires the SNARE VTI12 but not its close homologue, the conditionally redundant VTI11 protein. Furthermore, a vti12 mutant is specifically altered in transport of storage proteins, whereas a vti11 mutant is affected in transport of a lytic vacuole marker. These results demonstrate the specialization of VTI12 and VTI11 in mediating trafficking to storage and lytic vacuoles, respectively. Moreover, they validate the VAC2 secretion assay as a simple method to isolate genes that mediate trafficking to the PSV.