Molecular model for force production and transmission during vertebrate gastrulation.

Vertebrate embryos undergo dramatic shape changes at gastrulation that require locally produced and anisotropically applied forces, yet how these forces are produced and transmitted across tissues remains unclear. We show that depletion of myosin regulatory light chain (RLC) levels in the embryo blocks force generation at gastrulation through two distinct mechanisms: destabilizing the myosin II (MII) hexameric complex and inhibiting MII contractility. Molecular dissection of these two mechanisms demonstrates that normal convergence force generation requires MII contractility and we identify a set of molecular phenotypes correlated with both this failure of convergence force generation in explants and of blastopore closure in whole embryos. These include reduced rates of actin movement, alterations in C-cadherin dynamics and a reduction in the number of polarized lamellipodia on intercalating cells. By examining the spatial relationship between C-cadherin and actomyosin we also find evidence for formation of transcellular linear arrays incorporating these proteins that could transmit mediolaterally oriented tensional forces. These data combine to suggest a multistep model to explain how cell intercalation can occur against a force gradient to generate axial extension forces. First, polarized lamellipodia extend mediolaterally and make new C-cadherin-based contacts with neighboring mesodermal cell bodies. Second, lamellipodial flow of actin coalesces into a tension-bearing, MII-contractility-dependent node-and-cable actin network in the cell body cortex. And third, this actomyosin network contracts to generate mediolateral convergence forces in the context of these transcellular arrays.

Selective T-cell depletion targeting CD45RA reduces viremia and enhances early T-cell recovery compared with CD3-targeted T-cell depletion.

BACKGROUND: T-cell depletion (TCD) effectively reduces severe graft-versus-host disease in recipients of HLA-mismatched allografts. However, TCD is associated with delayed immune recovery and increased infections. We hypothesized that specific depletion of CD45RA+ naive T cells, rather than broad depletion of CD3+ T cells, can preserve memory-immunity in the allografts and confer protection against important viral infections in the early post-transplant period. METHODS: Sixty-seven patients who received TCD haploidentical donor transplantation for hematologic malignancy on 3 consecutive trials were analyzed. RESULTS: Patients receiving CD45RA-depleted donor grafts had 2000-fold more donor T cells infused, significantly higher T-cell counts at Day +30 post transplant (550/µL vs 10/µL; P < .001), and higher T-cell diversity by Vbeta spectratyping at Day +100 (P < .001). Importantly, these recipients experienced a significant reduction in both the incidence (P = .002) and duration (P = .02) of any viremia (cytomegalovirus, Epstein-Barr virus, or adenovirus) in the first 6 months post transplant. Specifically, recipients of CD3-depleted grafts were more likely to experience adenovirus viremia (27% vs 4%, P = .02). CONCLUSION: CD45RA-depletion provided a large number of donor memory T cells to the recipients and was associated with enhanced early T-cell recovery and protection against viremia.

Haploidentical stem cell transplantation augmented by CD45RA negative lymphocytes provides rapid engraftment and excellent tolerability.

BACKGROUND: Haploidentical donors are being increasingly used for allogeneic hematopoietic cell transplantation (HCT). However, the requisite T-cell depletion results in a profound and often long-lasting immunocompromised state, and donor lymphocyte infusions bring a risk of graft-versus-host disease (GVHD). Naïve T-cells are believed to be among the most alloreactive T-cell subset and can be identified by CD45RA expression. Allogeneic HCT using CD45RA depletion has not been previously described for haploidentical donors. PROCEDURE: Eight children with relapsed or refractory solid tumors were transplanted following myeloablative conditioning. Each patient received two cell products, one created by CD3 depletion and the other through CD45RA depletion. RESULTS: Median CD34 recovery was 59.2% with CD45RA depletion, compared to 82.4% using CD3 depletion. Median CD3+ T-cell dose after CD45RA reduction was 99.2 × 10(6) cells/kg, yet depletion of CD3+ CD45RA+ cells exceeded 4.5 log. CD45RA depletion also resulted in substantial depletion of B-cells (median 2.45 log). All eight patients engrafted within 14 days and rapidly achieved 100% donor chimerism. No acute GVHD or secondary graft failure was observed. CONCLUSIONS: CD45RA depletion is a novel approach to haploidentical HCT that offers rapid engraftment with minimal risk of GVHD.

Phase I study of the safety and pharmacokinetics of plerixafor in children undergoing a second allogeneic hematopoietic stem cell transplantation for relapsed or refractory leukemia.

The safety, pharmacokinetics, and biological effect of plerixafor in children as part of a conditioning regimen for chemo-sensitization in allogeneic hematopoietic stem cell transplantation (HSCT) have not been studied. This is a phase I study of plerixafor designed to evaluate its tolerability at dose of .24 mg/kg given intravenously on day -4 (level 1); day -4 and day -3 (level 2); or day -4, day -3, and day -2 (level 3) in combination with fludarabine, thiotepa, melphalan, and rabbit antithymocytic globulin for a second allogeneic HSCT in children with refractory or relapsed leukemia. Immunophenotype analysis was performed on blood and bone marrow before and after plerixafor administration. Twelve patients were enrolled. Plerixafor at all 3 levels was well tolerated without dose-limiting toxicity. Transient gastrointestinal side effects of National Cancer Institute-grade 1 or 2 in severity were the most common adverse events. The area under the concentration-time curve increased proportionally to the dose level. Plerixafor clearance was higher in males and increased linearly with body weight and glomerular filtration rate. The clearance decreased and the elimination half-life increased significantly from dose level 1 to 3 (P < .001). Biologically, the proportion of CXCR4(+) blasts and lymphocytes both in the bone marrow and peripheral blood increased after plerixafor administration.

Biomechanical properties of the capsule and extracellular matrix play a major role during the Wolffian/epididymal duct development.

BACKGROUND: The epididymis is important for sperm maturation and without its proper development, male infertility will result. Biomechanical properties of tissues/organs play key roles during their morphogenesis, including the Wolffian duct. It is hypothesized that structural/bulk stiffness of the capsule and mesenchyme/extracellular matrix that surround the duct is a major biomechanical property that regulates Wolffian duct morphogenesis. These data will provide key information as to the mechanisms that regulate the development of this important organ. OBJECTIVES: To measure the structural/bulk stiffness in Pascals (force/area) of the capsule and the capsule and mesenchyme together that surrounds the Wolffian duct during the development. To examine the relative membrane tension of mesenchymal cells during the Wolffian duct development. Since Ptk7 was previously shown to regulate ECM integrity and Wolffian duct elongation and coiling, the hypothesis that Ptk7 regulates structural/bulk stiffness and mesenchymal cell membrane tension was tested. MATERIALS AND METHODS: Atomic force microscopy and a microsquisher compression apparatus were used to measure the structural stiffness. Biomechanical properties within the membranes of cells within the capsule and mesenchyme were examined using a membrane-tension fluorescent probe. RESULTS AND DISCUSSION: The structural stiffness (Pascals) of the capsule and underlying mesenchyme was relatively constant during development, with a significant increase in the capsule at the later stages. However, this increase may reflect the ECM and associated mesenchyme being close to the capsule because the coiling of the duct pushed or compressed them into that space. Keeping the capsule and mesenchyme/ECM at constant stiffness would ensure that the duct will continue to coil under similar biomechanical forces throughout the development. Cells within the capsule and mesenchyme at different Wolffian duct regions during the development had varying degrees of membrane lipid tension. It is hypothesized that the dynamic changes ensure the duct is kept at a constant stiffness regardless of any external forces. Loss of Ptk7 resulted in an increase in stiffness at E18.5, which was presumable due to the loss of integrity of the ECM within the mesenchyme. CONCLUSION: Biomechanical properties of the capsule and the mesenchyme/extracellular matrix that surround the Wolffian duct play an important role toward Wolffian duct morphogenesis, thereby allowing for the proper development of the epididymis and subsequent male fertility.

Long-term outcome and evaluation of organ function in pediatric patients undergoing haploidentical and matched related hematopoietic cell transplantation for sickle cell disease.

HLA-matched related donor (MRD) hematopoietic stem cell transplantation (HSCT) is a well-established therapy for patients with sickle cell disease (SCD); however, experience using alternative donors, including haploidentical donors, in HSCT for SCD is limited. We report the long-term outcomes of 22 pediatric patients who underwent related donor HSCT for SCD at St. Jude Children's Research Hospital, either a myeloablative sibling MRD HSCT (n = 14) or reduced-intensity parental haploidentical donor HSCT (n = 8). The median patient age was 11.0 ± 3.9 years in the MRD graft recipients and 9.0 ± 5.0 years in the haploidentical donor graft recipients. The median follow-up was 9.0 ± 2.3 years, with an overall survival (OS) of 93% and a recurrence/graft failure rate of 0%, for the MRD cohort and 7.4 ± 2.4 years, with an OS of 75%, disease-free survival of 38%, and disease recurrence of 38%, for the haploidentical donor cohort. We report the long-term hematologic response and organ function in patients undergoing MRD or haploidentical donor HSCT for severe SCD. Our data demonstrate long-term hematologic improvements after HSCT with sustained engraftment, and confirm that HSCT offers long-term protection from common complications of SCD, including stroke, pulmonary hypertension, acute chest, and nephropathy, regardless of donor source.

Successful allogeneic hematopoietic cell engraftment after a minimal conditioning regimen in children with relapsed or refractory solid tumors.

Children with relapsed or refractory solid tumors face dismal prognoses, and novel therapies are desperately needed. Allogeneic hematopoietic cell transplantation (HCT) offers potential for cell-based therapy, but the toxicity of myeloablation limits this approach in heavily pretreated patients. We sought to determine the feasibility of HCT in a cohort of 24 children with incurable solid tumors using human leukocyte antigen-matched sibling or unrelated donors and a minimal conditioning regimen. Before stem cell infusion, all patients received 3 daily doses of 30 mg/m(2) fludarabine followed by 2 Gy of total body irradiation. Hematopoietic cell recovery was rapid and reliable. Median time to neutrophil engraftment was 13.5 days for sibling donors and 12 days for unrelated donors. Donor lymphocyte infusions were used safely in 4 patients, all of whom had either improved chimerism or apparent tumor response. Graft-versus-host disease was comparable across donor sources and did not affect survival. Relapse remains a substantial barrier, although objective graft-versus-tumor effect was observed in several patients. Four patients with detectable disease before HCT achieved a complete response for at least 30 days after HCT, and two remain long-term survivors. Three patients were in complete response before HCT and remained in remission for 3, 6, and 74 months after HCT. Early disease response was associated with improved survival. Allogeneic HCT using this conditioning regimen offers a potential platform for novel immunotherapies.

Modeling the roles of cohesotaxis, cell-intercalation, and tissue geometry in collective cell migration of Xenopus mesendoderm.

Collectively migrating Xenopus mesendoderm cells are arranged into leader and follower rows with distinct adhesive properties and protrusive behaviors. In vivo, leading row mesendoderm cells extend polarized protrusions and migrate along a fibronectin matrix assembled by blastocoel roof cells. Traction stresses generated at the leading row result in the pulling forward of attached follower row cells. Mesendoderm explants removed from embryos provide an experimentally tractable system for characterizing collective cell movements and behaviors, yet the cellular mechanisms responsible for this mode of migration remain elusive. We introduce an agent-based computational model of migrating mesendoderm in the Cellular-Potts computational framework to investigate the relative contributions of multiple parameters specific to the behaviors of leader and follower row cells. Sensitivity analyses identify cohesotaxis, tissue geometry, and cell intercalation as key parameters affecting the migration velocity of collectively migrating cells. The model predicts that cohesotaxis and tissue geometry in combination promote cooperative migration of leader cells resulting in increased migration velocity of the collective. Radial intercalation of cells towards the substrate is an additional mechanism to increase migratory speed of the tissue. Summary Statement: We present a novel Cellular-Potts model of collective cell migration to investigate the relative roles of cohesotaxis, tissue geometry, and cell intercalation on migration velocity of Xenopus mesendoderm.

Characterization of convergent thickening, a major convergence force producing morphogenic movement in amphibians.

The morphogenic process of convergent thickening (CT) was originally described as the mediolateral convergence and radial thickening of the explanted ventral involuting marginal zone (IMZ) of Xenopus gastrulae (Keller and Danilchik, 1988). Here, we show that CT is expressed in all sectors of the pre-involution IMZ, which transitions to expressing convergent extension (CE) after involution. CT occurs without CE and drives symmetric blastopore closure in ventralized embryos. Assays of tissue affinity and tissue surface tension measurements suggest CT is driven by increased interfacial tension between the deep IMZ and the overlying epithelium. The resulting minimization of deep IMZ surface area drives a tendency to shorten the mediolateral (circumblastoporal) aspect of the IMZ, thereby generating tensile force contributing to blastopore closure (Shook et al., 2018). These results establish CT as an independent force-generating process of evolutionary significance and provide the first clear example of an oriented, tensile force generated by an isotropic, Holtfreterian/Steinbergian tissue affinity change.

Progenitor cell therapy for acquired pediatric nervous system injury: Traumatic brain injury and acquired sensorineural hearing loss.

While cell therapies hold remarkable promise for replacing injured cells and repairing damaged tissues, cell replacement is not the only means by which these therapies can achieve therapeutic effect. For example, recent publications show that treatment with varieties of adult, multipotent stem cells can improve outcomes in patients with neurological conditions such as traumatic brain injury and hearing loss without directly replacing damaged or lost cells. As the immune system plays a central role in injury response and tissue repair, we here suggest that multipotent stem cell therapies achieve therapeutic effect by altering the immune response to injury, thereby limiting damage due to inflammation and possibly promoting repair. These findings argue for a broader understanding of the mechanisms by which cell therapies can benefit patients.

Autologous hematopoietic cell transplantation for the treatment of relapsed/refractory pediatric, adolescent, and young adult Hodgkin lymphoma: a single institutional experience.

Pediatric, adolescent, and young adult patients with relapsed or refractory Hodgkin lymphoma receive multimodal therapy, including autologous hematopoietic cell transplantation (AutoHCT). Despite aggressive therapy, historical outcomes for this patient population have been poor. This paper describes a single institutional experience utilizing AutoHCT in 74 patients treated from 1988-2015. Our results demonstrate significantly improved outcomes over time. Compared with patients treated in the earlier era (1988-2001), 5-year overall survival improved from 62.5 ± 9.6% to 91.8 ± 4.4% (p < 0.001) and event free survival improved from 41.7 ± 9.6% to 87.7 ± 5.3% (I < 0.001) for patients treated in a later era (2002-2015). Improvements in survival are multifactorial, including reductions in both relapse and nonrelapse mortality. Further investigation is needed to determine the role of AutoHCT in a modern treatment cohort that includes frequent use of targeted immunotherapies. In addition, as the use and availability of effective novel therapeutics increases for this patient population there may be an opportunity for the reduction of standard cytotoxic therapies, including in AutoHCT preparative regimens, thereby mitigating late effects.

Large, long range tensile forces drive convergence during Xenopus blastopore closure and body axis elongation.

Indirect evidence suggests that blastopore closure during gastrulation of anamniotes, including amphibians such as Xenopus laevis, depends on circumblastoporal convergence forces generated by the marginal zone (MZ), but direct evidence is lacking. We show that explanted MZs generate tensile convergence forces up to 1.5 µN during gastrulation and over 4 µN thereafter. These forces are generated by convergent thickening (CT) until the midgastrula and increasingly by convergent extension (CE) thereafter. Explants from ventralized embryos, which lack tissues expressing CE but close their blastopores, produce up to 2 µN of tensile force, showing that CT alone generates forces sufficient to close the blastopore. Uniaxial tensile stress relaxation assays show stiffening of mesodermal and ectodermal tissues around the onset of neurulation, potentially enhancing long-range transmission of convergence forces. These results illuminate the mechanobiology of early vertebrate morphogenic mechanisms, aid interpretation of phenotypes, and give insight into the evolution of blastopore closure mechanisms.

Mechanical strain determines the axis of planar polarity in ciliated epithelia.

Epithelia containing multiciliated cells align beating cilia along a common planar axis specified by the conserved planar cell polarity (PCP) pathway. Specification of the planar axis is also thought to require a long-range cue to align the axis globally, but the nature of this cue in ciliated and other epithelia remains poorly understood. We examined this issue using the Xenopus larval skin, where ciliary flow aligns to the anterior-posterior (A-P) axis. We first show that a planar axis initially arises in the developing skin during gastrulation, based on the appearance of polarized apical microtubules and cell junctions with increased levels of stable PCP components. This axis also arises in severely ventralized embryos, despite their deficient embryonic patterning. Because ventralized embryos still gastrulate, producing a mechanical force that strains the developing skin along the A-P axis, we asked whether this strain alone drives global planar patterning. Isolated skin explanted before gastrulation lacks strain and fails to acquire a global planar axis but responds to exogenous strain by undergoing cell elongation, forming polarized apical microtubules, and aligning stable components of the PCP pathway orthogonal to the axis of strain. The planar axis in embryos can be redirected by applying exogenous strain during a critical period around gastrulation. Finally, we provide evidence that apical microtubules and the PCP pathway interact to align the planar axis. These results indicate that oriented tissue strain generated by the gastrulating mesoderm plays a major role in determining the global axis of planar polarity of the developing skin.

Monitoring Central Venous Catheter Resistance to Predict Imminent Occlusion: A Prospective Pilot Study.

BACKGROUND: Long-term central venous catheters are essential for the management of chronic medical conditions, including childhood cancer. Catheter occlusion is associated with an increased risk of subsequent complications, including bloodstream infection, venous thrombosis, and catheter fracture. Therefore, predicting and pre-emptively treating occlusions should prevent complications, but no method for predicting such occlusions has been developed. METHODS: We conducted a prospective trial to determine the feasibility, acceptability, and efficacy of catheter-resistance monitoring, a novel approach to predicting central venous catheter occlusion in pediatric patients. Participants who had tunneled catheters and were receiving treatment for cancer or undergoing hematopoietic stem cell transplantation underwent weekly catheter-resistance monitoring for up to 12 weeks. Resistance was assessed by measuring the inline pressure at multiple flow-rates via a syringe pump system fitted with a pressure-sensing transducer. When turbulent flow through the device was evident, resistance was not estimated, and the result was noted as "non-laminar." RESULTS: Ten patients attended 113 catheter-resistance monitoring visits. Elevated catheter resistance (>8.8% increase) was strongly associated with the subsequent development of acute catheter occlusion within 10 days (odds ratio = 6.2; 95% confidence interval, 1.8-21.5; p <0.01; sensitivity, 75%; specificity, 67%). A combined prediction model comprising either change in resistance greater than 8.8% or a non-laminar result predicted subsequent occlusion (odds ratio = 6.8; 95% confidence interval, 2.0-22.8; p = 0.002; sensitivity, 80%; specificity, 63%). Participants rated catheter-resistance monitoring as highly acceptable. CONCLUSIONS: In this pediatric hematology and oncology population, catheter-resistance monitoring is feasible, acceptable, and predicts imminent catheter occlusion. Larger studies are required to validate these findings, assess the predictive value for other clinical outcomes, and determine the impact of pre-emptive therapy. TRIAL REGISTRATION: Clinicaltrials.gov NCT01737554.

Longitudinal changes in body mass and composition in survivors of childhood hematologic malignancies after allogeneic hematopoietic stem-cell transplantation.

PURPOSE: To measure longitudinal changes in body mass and composition in survivors of childhood hematologic malignancies after allogeneic hematopoietic stem-cell transplantation (HSCT). PATIENTS AND METHODS: Body mass index (BMI) was analyzed in 179 survivors by category (underweight, healthy-weight, overweight, and obese) and by z score. Fat and lean body mass measured by dual-energy x-ray absorptiometry was analyzed as z scores. RESULTS: Over a median 6.6 years of follow-up, BMI z scores diminished significantly (0.32 pre-HSCT v -0.60 at 10 years post-HSCT; P < .001). Mean z scores for fat mass stayed within population norms, but those for lean mass remained below normal levels and diminished significantly over time (P = .018). Pre-HSCT BMI category and/or z score were strongly predictive of post-HSCT BMI (P < .001) and of fat and lean mass z scores (both P < .001). Survivors with extensive chronic graft-versus-host disease were more likely than others to have low BMI (P = .004) and low lean mass (P < .001) post-HSCT. Older age at HSCT (P = .015) and T-cell-depleted graft (P = .018) were predictive of lower post-HSCT BMI. Female patients had higher body fat (P = .002) and lower lean mass (P = .013) z scores than male patients, and black patients had higher fat mass z scores than white patients (P = .026). CONCLUSION: BMI declines significantly after allogeneic HSCT for childhood hematologic malignancies, reflecting primarily a substantial decrease in lean mass but not fat mass. Monitoring and preservation of BMI and lean mass are vital, especially in those with the identified risk factors.

Cytotoxicity of activated natural killer cells against pediatric solid tumors.

PURPOSE: To develop new therapies for children with solid tumors, we tested the cytotoxicity of natural killer (NK) cells expanded by coculture with K562-mb15-41BBL cells. We sought to identify the most sensitive tumor subtypes, clarify the molecular interactions regulating cytotoxicity, and determine NK antitumor potential in vivo. EXPERIMENTAL DESIGN: We tested in vitro cytotoxicity of expanded NK cells against cell lines representative of Ewing sarcoma (EWS; n = 5), rhabdomyosarcoma (n = 4), neuroblastoma (n = 3), and osteosarcoma (n = 3), and correlated the results with expression of inhibitory and activating NK receptor ligands. We also compared expanded and primary NK cells, determined the effects of activating receptor ligation and of chemotherapeutic drugs, and assessed the therapeutic effect of NK cell infusions in xenografts. RESULTS: In 45 experiments, EWS and rhabdomyosarcoma cell lines were remarkably sensitive to expanded NK cells, with median cytotoxicities at 1:1 effector/target ratio of 87.2% and 79.1%, respectively. Cytotoxicity was not related to levels of expression of NK receptor ligands, nor was it affected by pretreatment of target cells with daunorubicin or vincristine, but was markedly inhibited by preincubation of NK cells with a combination of antibodies against the NK-activating receptors NKGD2 and DNAM-1. Expanded NK cells were considerably more cytotoxic than unstimulated NK cells, and eradicated EWS cells engrafted in nonobese diabetic/severe combined immunodeficient Il2rgnull mice. CONCLUSIONS: Among pediatric solid tumors, EWS and rhabdomyosarcoma are exquisitely sensitive to expanded NK cells. The NK expansion method described here has been adapted to large-scale conditions and supports a phase I clinical study including patients with these malignancies.

Morphogenic machines evolve more rapidly than the signals that pattern them: lessons from amphibians.

The induction of mesoderm and the patterning of its dorsal-ventral and anterior-posterior axes seems to be relatively conserved throughout the chordates, as do the morphogenic movements that produce a phylotypic stage embryo. What is not conserved is the initial embryonic architecture of the fertilized egg, and the specific cell behaviors used to drive mesoderm morphogenesis. How then do conserved patterning pathways adapt to diverse architectures and where do they diverge to direct the different cell behaviors used to shape the phylotypic body plan? Amphibians in particular, probably because of their broad range of reproductive strategies, show diverse embryonic architectures across their class and use diverse cell behaviors during their early morphogenesis, making them an interesting comparative group. We examine three examples from our work on amphibians that show variations in the use of cell behaviors to drive the morphogenesis of the same tissues. We also consider possible points where the conserved patterning pathways might diverge to produce different cell behaviors.

Epithelial type, ingression, blastopore architecture and the evolution of chordate mesoderm morphogenesis.

Chordate embryos show an evolutionary trend in the mechanisms they use to internalize presumptive mesoderm, relying predominantly on invagination in the basal chordates, varying combinations of involution and ingression in the anamniote vertebrates and reptiles, and predominantly on ingression in birds and mammals. This trend is associated with variations in epithelial type and changes in embryonic architecture as well as variations in the type of blastopore formed by an embryo. We also note the surprising conservation of the involution, during gastrulation, of at least a subset of the notochordal cells throughout the chordates, and suggest that this indicates a constraint on morphogenic evolution based on a functional linkage between architecture and patterning. Finally, we propose a model for the evolutionary transitions from gastrulation through a urodele amphibian-type blastopore to gastrulation through a primitive streak, as in chick or mouse.

How we are shaped: the biomechanics of gastrulation.

Although it is rarely considered so in modern developmental biology, morphogenesis is fundamentally a biomechanical process, and this is especially true of one of the first major morphogenic transformations in development, gastrulation. Cells bring about changes in embryonic form by generating patterned forces and by differentiating the tissue mechanical properties that harness these forces in specific ways. Therefore, biomechanics lies at the core of connecting the genetic and molecular basis of cell activities to the macroscopic tissue deformations that shape the embryo. Here we discuss what is known of the biomechanics of gastrulation, primarily in amphibians but also comparing similar morphogenic processes in teleost fish and amniotes, and selected events in several species invertebrates. Our goal is to review what is known and identify problems for further research.