Biomechanical analysis of progressive rotator cuff tendon tears on superior stability of the shoulder.

BACKGROUND: The biomechanical relationship between irreparable rotator cuff tear size and glenohumeral joint stability in the setting of superiorly directed forces has not been characterized. The purpose of this study was to quantify kinematic alterations of the glenohumeral joint in response to superiorly directed forces in a progressive posterosuperior rotator cuff tear model. METHODS: Nine fresh-frozen cadaveric shoulders (mean age; 58 years) were tested with a custom shoulder testing system. Three conditions were tested: intact, stage II (supraspinatus) tear, stage III (supraspinatus + anterior half of infraspinatus) tear. At each condition, range of motion and humeral head positions were measured with a "balanced" loading condition, and with a superiorly directed force ("unbalanced loading condition"). At each of the 0°, 20°, and 40° of glenohumeral abduction positions, all measurements were made at 0°, 30°, 60°, and 90° of external rotation (ER). Two-way repeated measures analysis of variance with Tukey post hoc tests were performed for statistical analyses. RESULTS: With the balanced load, no significant change in superior humeral head position was observed in stage II tears. Stage III tears significantly changed the humeral head position superiorly at 30° and 60° ER at each abduction angle compared with the intact condition (P ≤ .028). With superiorly directed load, stage II and stage III tears both showed statistically significant increases in superior translation at all degrees of ER for all degrees of abduction (P ≤ .035), except stage II tears at 0° ER and 40° abduction (P = .185) compared with the intact condition. Stage II tears showed posterior translations with 30° and 60° ER, both at 20° and 40° of abduction. Stage III tears also showed posterior translations with 90° ER for all abduction angles (P ≤ .039). CONCLUSION: With superiorly directed loads, complete supraspinatus tendon tears created superior translations at all abduction angles, and posterior instability in the middle ranges of rotation for 20° and 40° of abduction. Larger tears involving the anterior half of the infraspinatus tendon caused significant superior and posterior translations within the middle ranges of ER for all abduction angles. In addition to superior instability, posterior translation should be considered when selecting or developing surgical techniques for large posterosuperior rotator cuff tears.

Anterior Cable Reconstruction Using the Proximal Biceps Tendon for Large Rotator Cuff Defects Limits Superior Migration and Subacromial Contact Without Inhibiting Range of Motion: A Biomechanical Analysis.

PURPOSE: To assess an anterior cable reconstruction (ACR) using autologous proximal biceps tendon for large to massive rotator cuff tears. METHODS: Nine cadaveric shoulders (mean age, 58 years) were tested with a custom testing system. Range of motion, superior translation of the humeral head, and subacromial contact pressure were measured at 0°, 30°, 60°, and 90° of external rotation (ER) with 0°, 20°, and 40° of glenohumeral abduction. Five conditions were tested: intact, stage II tear (supraspinatus), stage II tear + ACR, stage III tear (supraspinatus + anterior half of infraspinatus), and stage III tear + ACR. ACR involved a biceps tendon tenotomy at the transverse humeral ligament, preserving its labral attachment. ACR included nonpenetrating suture-loop fixation using 2 side-to-side sutures and an anchor at the articular margin to restore anatomy and secure the tendon along the anterior edge of the cuff defect. ACR was performed in 20° glenohumeral abduction and 60° ER. RESULTS: ACR for both stage II and stage III showed significantly higher total range of motion compared with intact at all angles (P ≤ .001). ACR significantly decreased superior translation for stage II tears at 0°, 30°, and 60° ER for both 0° and 20° abduction (P ≤ .01) and for stage III tears at 0° and 30° ER for both 0° and 20° abduction (P ≤ .004). ACR for stage III tear significantly reduced peak subacromial contact pressure at 30° and 60° ER with 0° and 40° abduction and at 30° ER with 20° abduction (P ≤ .041). CONCLUSIONS: ACR using autologous biceps tendon biomechanically normalized superior migration and subacromial contact pressure, without limiting range of motion. CLINICAL RELEVANCE: ACR may improve rotator cuff tendon repair longevity by providing basic static ligamentous support to the dynamic tendon while helping to limit superior migration without restricting glenohumeral kinematics.

Knotless Transosseous-Equivalent Rotator Cuff Repair Improves Biomechanical Self-reinforcement Without Diminishing Footprint Contact Compared With Medial Knotted Repair.

PURPOSE: To assess the effect of medial-row knots on self-reinforcement and footprint contact characteristics for transosseous-equivalent repair compared with the same construct without knots. METHODS: In 8 fresh-frozen human shoulders, transosseous-equivalent repairs with and without medial-row mattress knots were performed in each specimen. A pressure sensor was fixed at the tendon-footprint interface for all repairs. Parameters measured included footprint contact area, force, and pressure. The supraspinatus tendon was loaded sequentially from 0 to 60 N at 0° and 30° of abduction. RESULTS: Both repairs provided a linear progression (slope) of footprint force and pressure as increasing tendon loads were applied. However, the knotless repair had a significantly higher progression ("self-reinforcement" effect) than the knotted repair at both abduction angles (P = .006 at 0° and P = .021 at 30°). The addition of medial-row knots did not significantly change the footprint contact area (in square millimeters), contact force (in newtons), contact pressure (in kilopascals), or peak pressure (in kilopascals) at each load tested, as well as at both abduction angles. For a given repair, only the knotless repair had significant decreases in contact area, contact force, contact pressure, and peak pressure with increasing abduction angles from 0° to 30° (P = .004 and P = .048). CONCLUSIONS: Knotless transosseous-equivalent repair shows an improved self-reinforcement effect, without diminishing footprint contact, compared with the same repair with medial knots. Although knotless repair itself can show diminished footprint contact with abduction, medial knots show an adverse biomechanical effect by inhibiting self-reinforcement, without improving contact characteristics compared with knotless repair at each abduction angle tested. Clinical outcomes with specific indications, on the basis of these findings, require further investigation. CLINICAL RELEVANCE: This study biomechanically helps to validate studies that have shown clinical success with knotless transosseous-equivalent repair. The inhibition of self-reinforcement may provide a quantified biomechanical rationale for medial tear patterns seen with knotted repairs.

Biomechanical effects of a 2 suture-pass medial inter-implant mattress on transosseous-equivalent rotator cuff repair and considerations for a "technical efficiency ratio".

BACKGROUND: Rotator cuff repair involving fewer tendon suture passes without compromising biomechanical performance would represent a technical advancement. An inter-implant "medial pulley-mattress" transosseous-equivalent (MP-TOE) repair requiring fewer tendon suture-passes was hypothesized to provide equivalent biomechanical characteristics compared to the control. METHODS: In 6 human cadaveric shoulders, a transosseous-equivalent (TOE) repair (control) was performed utilizing 2 separate medial mattresses resulting in 4 tendon-bridging sutures. In 6 matched-pairs, 2 single-loaded anchors were used to create a medial inter-implant mattress construct (all sutures shuttled in 1 tendon pass per anchor)-after knot-tying, the same tendon-bridging pattern as the control was created. A materials testing machine cyclically loaded each repair from 10-180 N for 30 cycles; each repair subsequently underwent failure testing. Gap and strain were measured with a video digitizing system. A "technical efficiency ratio" (TER) was defined as: (#knots + #suture passes + #suture limbs)/#fixation points. RESULTS: Cyclic and failure testing demonstrated no significant differences between constructs. Gap formation at cycle 30 was 5.3 ± 0.8 mm (TOE) and 5.0 ± 0.3 mm (MP-TOE) (P = .62). Cycle 30 anterior strain values were -16.0 ± 7.3% (TOE) and -15.8 ± 6.6% (MP-TOE) (P = .99). Yield loads were 208.7 ± 2.7 N (TOE) and 204.0 ± 1.3 N (MP-TOE) (P = .17). Mode of failure demonstrated less tendon cut-out with the MP-TOE repair. The MP-TOE repair has a TER of 2.0 vs 2.5 for the control. CONCLUSION: The MP-TOE repair requiring fewer tendon suture passes, yet creating an additional inter-implant mattress configuration, is biomechanically equivalent to the original TOE technique, and may limit failure with improved medial load-sharing capacity. A TER may help quantify technical ease and help standardize comparisons between repair techniques.

Does transosseous-equivalent rotator cuff repair biomechanically provide a "self-reinforcement" effect compared with single-row repair?

BACKGROUND: Transosseous-equivalent (TOE) rotator cuff repair has been theorized to be "self-reinforcing" against potentially destructive and increasing tendon loads. The goal of this study was to biomechanically verify and characterize the effect of increasing tendon load on frictional resistance over a repaired footprint for single-row (SR) and TOE repair techniques. METHODS: In 10 fresh frozen human shoulders, TOE and SR supraspinatus tendon repairs were performed in each specimen. For all repairs, a pressure sensor was secured at the tendon-footprint interface. The supraspinatus tendon was loaded with 0, 20, 40, 60, and 80 N at 0° and 30° abduction. Paired t tests and multivariate regression analyses were used for comparisons. RESULTS: The SR repair had significant increases in footprint contact force, area, and pressure between each and all tendon-loading conditions (P < .05). The TOE repair similarly demonstrated increases in footprint contact force with increasing tendon load (P < .05). Comparing between repairs, TOE repair had more footprint contact force, area, pressure, and peak pressure at each load for both abduction angles (P < .05). With increasing load, the TOE repair had a significantly higher progression (slope) of footprint force and pressure compared with the SR repair. CONCLUSIONS: Self-reinforcing capacity in rotator cuff repair has been biomechanically characterized and verified. The TOE repair, with tendon-bridging sutures fixed medially and spanning the footprint, provides disproportionately more progressive footprint frictional resistance with increasing tendon loads compared with the SR repair secured over isolated fixation points. This self-reinforcing effect could help sustain structural integrity and potentially improve healing biology.

Rotator cuff tendon repair morphology comparing 2 single-anchor repair techniques.

PURPOSE: To compare the effect of 2 common rotator cuff repair techniques, for smaller tears limited to the use of a single anchor, on tendon morphology in relation to the footprint. METHODS: Six matched pairs of human shoulders were dissected, and a standardized 10-mm supraspinatus tendon tear was created. Two single-anchor repairs were performed: simple repair with the anchor on the footprint or inverted-mattress repair with the anchor 1 cm distal-lateral to the footprint. The repaired specimens were frozen in situ with liquid nitrogen. Coronal cross sections through the intact and repaired tendon were made. A digitizer was used to measure variables including tendon area and radius of tendon curvature. RESULTS: Comparing between repairs, we found significantly more gap formation for the simple repair at the repair cross section (3.67 ± 0.32 mm v 0.68 ± 0.10 mm, P = .00050). The simple repair had less tendon area (38.28 ± 2.50 mm(2)v 58.65 ± 4.06 mm(2), P = .0036) and a smaller radius of curvature (8.47 ± 1.39 mm v 32.51 ± 3.94 mm, P = .0046). For the simple repair, there was significantly more gap formation, less tendon area, and a smaller radius of tendon curvature for all repair cross sections compared with the intact cross sections (P < .05). For the inverted-mattress repair, there was more gap formation compared with the intact condition (P < .05), although it was less than 1 mm on average; for tendon area, radius of curvature, and tendon height, the cross section centered on the repair showed no differences compared with the intact control. CONCLUSIONS: For rotator cuff tears that are 10 mm or smaller and limited to the use of a single anchor, using a distal-lateral anchor position with tape-type suture can provide better maintenance of native tendon morphology and footprint dimensions when compared with repair that uses standard sutures and places the anchor on the footprint. CLINICAL RELEVANCE: For smaller tears, the inverted-mattress repair described in this article may provide a relatively improved healing environment compared with a simple repair on the footprint, potentially optimizing the prevention of early tear progression.

Requirement for the budding yeast polo kinase Cdc5 in proper microtubule growth and dynamics.

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of the budding yeast polo kinase Cdc5, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal noncatalytic polo box domain, a region that is critical for proper subcellular localization. One of these mutants, cdc5-11, exhibited a temperature-sensitive growth defect with an abnormal spindle morphology. Strikingly, provision of a moderate level of benomyl, a microtubule-depolymerizing drug, permitted cdc5-11 cells to grow significantly better than the isogenic CDC5 wild type in a FEAR (cdc Fourteen Early Anaphase Release)-independent manner. In addition, cdc5-11 required MAD2 for both cell growth and the benomyl-remedial phenotype. These results suggest that cdc5-11 is defective in proper spindle function. Consistent with this view, cdc5-11 exhibited abnormal spindle morphology, shorter spindle length, and delayed microtubule regrowth at the nonpermissive temperature. Overexpression of CDC5 moderately rescued the spc98-2 growth defect. Interestingly, both Cdc28 and Cdc5 were required for the proper modification of the spindle pole body components Nud1, Slk19, and Stu2 in vivo. They also phosphorylated these three proteins in vitro. Taken together, these observations suggest that concerted action of Cdc28 and Cdc5 on Nud1, Slk19, and Stu2 is important for proper spindle functions.

Beta1 integrin inhibitory antibody induces apoptosis of breast cancer cells, inhibits growth, and distinguishes malignant from normal phenotype in three dimensional cultures and in vivo.

Current therapeutic approaches to cancer are designed to target molecules that contribute to malignant behavior but leave normal tissues intact. beta(1) integrin is a candidate target well known for mediating cell-extracellular matrix (ECM) interactions that influence diverse cellular functions; its aberrant expression has been implicated in breast cancer progression and resistance to cytotoxic therapy. The addition of beta(1) integrin inhibitory agents to breast cancer cells at a single-cell stage in a laminin-rich ECM (three-dimensional lrECM) culture was shown to down-modulate beta(1) integrin signaling, resulting in malignant reversion. To investigate beta(1) integrin as a therapeutic target, we modified the three-dimensional lrECM protocol to approximate the clinical situation: before treatment, we allowed nonmalignant cells to form organized acinar structures and malignant cells to form tumor-like colonies. We then tested the ability of beta(1) integrin inhibitory antibody, AIIB2, to inhibit tumor cell growth in several breast cancer cell lines (T4-2, MDA-MB-231, BT474, SKBR3, and MCF-7) and one nonmalignant cell line (S-1). We show that beta(1) integrin inhibition resulted in a significant loss of cancer cells, associated with a decrease in proliferation and increase in apoptosis, and a global change in the composition of residual colonies. In contrast, nonmalignant cells that formed tissue-like structures remained resistant. Moreover, these cancer cell-specific antiproliferative and proapoptotic effects were confirmed in vivo with no discernible toxicity to animals. Our findings indicate that beta(1) integrin is a promising therapeutic target, and that the three-dimensional lrECM culture assay can be used to effectively distinguish malignant and normal tissue response to therapy.

Novel functional dissection of the localization-specific roles of budding yeast polo kinase Cdc5p.

Budding yeast polo kinase Cdc5p localizes to the spindle pole body (SPB) and to the bud-neck and plays multiple roles during M-phase progression. To dissect localization-specific mitotic functions of Cdc5p, we tethered a localization-defective N-terminal kinase domain of Cdc5p (Cdc5pDeltaC) to the SPB or to the bud-neck with components specifically localizing to one of these sites and characterized these mutants in a cdc5Delta background. Characterization of a viable, SPB-localizing, CDC5DeltaC-CNM67 mutant revealed that it is defective in timely degradation of Swe1p, a negative regulator of Cdc28p. Loss of BFA1, a negative regulator of mitotic exit, rescued the lethality of a neck-localizing CDC5DeltaC-CDC12 or CDC5DeltaC-CDC3 mutant but yielded severe defects in cytokinesis. These data suggest that the SPB-associated Cdc5p activity is critical for both mitotic exit and cytokinesis, whereas the bud neck-localized Cdc5p is required for proper Swe1p regulation. Interestingly, a cdc5Delta bfa1Delta swe1Delta triple mutant is viable but grows slowly, whereas cdc5Delta cells bearing both CDC5DeltaC-CNM67 and CDC5DeltaC-CDC12 grow well with only a mild cell cycle delay. Thus, SPB- and the bud-neck-localized Cdc5p control most of the critical Cdc5p functions and downregulation of Bfa1p and Swe1p at the respective locations are two critical factors that require Cdc5p.

Bni5p, a septin-interacting protein, is required for normal septin function and cytokinesis in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Delta strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Delta mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.

Requirement for Bbp1p in the proper mitotic functions of Cdc5p in Saccharomyces cerevisiae.

The polo-box domain of the budding yeast polo kinase Cdc5p plays an essential role for targeting the catalytic activity of Cdc5p to spindle pole bodies (SPBs) and cytokinetic neck-filaments. Here, we report the isolation of Bbp1p as a polo-box interacting protein by a yeast two-hybrid screen. Bbp1p localizes to the periphery of the central plaque of the SPB and plays an important role in SPB duplication. Similarly, Cdc5p localized to the cytoplasmic periphery of the SPB. In vitro binding studies showed that Cdc5p interacted with the N-terminal domain of Bbp1p (Bbp1pDeltaC), but apparently not with Mps2p, a component shown to form a stable complex with Bbp1p. In addition, Bbp1p, but likely not Mps2p, was required for proper localization of Cdc5p to the SPB. The C-terminal coiled-coil domain of Bbp1p (Bbp1p(243-385)), which is crucial for both the homodimerization and the SPB localization, could target the localization-defective Cdc5pDeltaC to the SPB and induce the release of Cdc14p from the nucleolus. Consistent with this observation, expression of CDC5DeltaC-BBP1(243-385) under CDC5 promoter control partially complemented the cdc5Delta defect. These data suggest that Bbp1pDeltaC interacts with the polo-box domain of Cdc5p, and this interaction is critical for the subcellular localization and mitotic functions of Cdc5p.

Yeast polo-like kinases: functionally conserved multitask mitotic regulators.

The polo-like kinases (Plks) are a conserved subfamily of Ser/Thr protein kinases that play pivotal roles in regulating various cellular and biochemical events at multiple stages of M phase. Genetic and biochemical data revealed that both the budding yeast and the fission yeast polo kinase homologs (Cdc5 and Plo1, respectively) bear remarkable functional similarities with those in metazoan organisms, suggesting that the role of Plks is largely conserved throughout evolution. Thus, studies on Plks in genetically amenable lower eucaryotic organisms may yield valuable insights into the function of Plks in higher eucaryotic organisms. In this review, common properties and distinct functions of Cdc5 and Plo1 will be discussed and compared to properties and functions of Plks in higher eucaryotic organisms.

Biomechanical analysis of a knotless transtendon interimplant mattress repair for partial-thickness articular-sided rotator cuff tears.

BACKGROUND: A transtendon interimplant mattress repair along the medial row for partial-thickness rotator cuff repairs has been described with clinical success. However, the biomechanical characteristics for such a repair have not been elucidated. HYPOTHESIS: A knotless interimplant mattress repair may show improved or equivalent load and strain characteristics, compared with a repair using isolated mattress repairs over each of 2 anchors. STUDY DESIGN: Controlled laboratory study. METHODS: Seven matched pairs of human cadaveric shoulders were dissected. Articular-sided tears were created involving 50% of the supraspinatus footprint. In 7 shoulders, repairs were performed with mattress configurations isolated over each of 2 anchor sites (control group). In 7 contralateral shoulders, a knotless interimplant mattress suture configuration was employed creating bridging sutures between implants. For all specimens, a materials-testing machine was used to cyclically load each repair from 10 to 180 N for 30 cycles; each repair was then loaded to failure. A deformation rate of 1 mm per second was employed for all tests. A video-digitizing system was employed to quantitatively measure the gap formation and strain on the footprint area of the repair. For detecting gap formation, 7 matched pairs were necessary for achieving a power of at least 90%. RESULTS: During cyclic loading, gap formation at the anterior tendon was significantly lower in the control group (P < .05) but did not exceed 0.5 mm. There were no significant differences for linear stiffness, hysteresis, and strain between the 2 constructs. During tensile load-to-failure testing, there were no significant differences at yield load between the control and knotless techniques (293.90 + or - 132.72 N and 320.38 + or - 237.01 N, respectively; P > .05). There were no differences for stiffness, ultimate load, and energy absorbed to failure between the 2 repairs (P > .05). Gap formation in 3 regions was not significantly different between groups at yield and ultimate loads (P > .05). The anterior regions of the repair were the first to fail in all constructs. CONCLUSION: A transtendon interimplant mattress rotator cuff repair for partial articular-sided tendon tears involving 50% of the footprint has biomechanical characteristics similar to those of a repair employing 2 isolated mattress configurations. An interim-plant mattress repair can protect tendon strain; it also exhibits yield loads that exceed those typically experienced in the early postoperative period. CLINICAL RELEVANCE: A medial-row interimplant mattress repair configuration that is knotless may facilitate repair without compromising biomechanical characteristics.

The effect of abduction and rotation on footprint contact for single-row, double-row, and modified double-row rotator cuff repair techniques.

BACKGROUND: An abduction pillow and abduction and rotation exercises are commonly used after rotator cuff repair. The effect of glenohumeral abduction and rotation on footprint contact has not been elucidated. HYPOTHESIS: Abduction will decrease tendon-to-bone contact for all repairs. A modified double-row repair will maintain footprint contact more effectively at each position of humeral abduction and rotation than double- or single-row repairs. STUDY DESIGN: Controlled laboratory study. METHODS: In 6 fresh-frozen human shoulders, a modified double-row supraspinatus tendon repair was performed; a suture limb from each of 2 medial anchors was bridged over the tendon and fixed laterally. Double- and single-row repairs were performed sequentially; a total of 3 repairs were tested. For all repairs, a Tekscan pressure sensor was fixed at the tendon-footprint interface. The tendon was loaded with 30 N. The shoulders were tested at 0 degrees , 30 degrees , and 60 degrees of abduction with 0 degrees of rotation. For both dual-row repairs, 5 rotation positions were tested. RESULTS: The greatest contact areas at neutral rotation were achieved at 0 degrees of abduction for the modified double-row, double-row, and single-row repairs (151.3 +/- 10.7 mm2, 80.7 +/- 30.0 mm2, and 61.3 +/- 26.1 mm2, respectively), with values decreasing as abduction increased. Each repair was significantly different from one another at each abduction angle (P < .05), except between single- and double-row repairs at 0 degrees of abduction. Mean interface pressure exerted over the footprint was greater for the modified double-row technique than for the other techniques at each abduction angle (P < .05). With respect to rotation, the modified double-row repair had significantly more footprint contact than did the double-row repair at each position tested (P < .05). CONCLUSION: For a given repair, increasing abduction at neutral rotation reduced footprint contact. Internal rotation to 60 degrees provided among the highest contact measurements. The modified double-row technique provided the most contact. CLINICAL RELEVANCE: Results are consistent with the practice of immobilizing the shoulder with 30 degrees or less of abduction and up to 60 degrees of internal rotation to optimize footprint contact. A dual-row repair may maximize contact when initiating rehabilitation that involves abduction and rotation.

Beta1 integrin inhibition dramatically enhances radiotherapy efficacy in human breast cancer xenografts.

Beta(1) integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of beta(1) integrin signaling. We previously showed that beta(1) integrin inhibitory antibodies (e.g., AIIB2) enhance apoptosis and decrease growth in human breast cancer cells in three-dimensional laminin-rich extracellular matrix (lrECM) cultures and in vivo. Here, we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used three-dimensional lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in three-dimensional lrECM. Colonies were assayed for apoptosis and beta(1) integrin/Akt signaling pathways were evaluated using Western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in three-dimensional lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down-regulating Akt in breast cancer colonies in three-dimensional lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared with either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We previously showed that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo.

The biomechanical effects of dynamic external rotation on rotator cuff repair compared to testing with the humerus fixed.

BACKGROUND: Biomechanical testing without humeral motion is a standard method for evaluating rotator cuff repair constructs. This cannot elucidate the effects of dynamic external rotation on the repair, which is a common postoperative motion. HYPOTHESIS: Biomechanical properties and gap formation of rotator cuff repairs will be different when dynamic external rotation is allowed to occur during loading. STUDY DESIGN: Controlled laboratory study. METHODS: In 6 matched pairs of human cadaveric shoulders, a commonly used single-row rotator cuff repair was performed. In 6 shoulders, a materials testing machine and a custom testing apparatus that permits cyclic rotation (0 degrees -30 degrees ) were employed (group 1). In contralateral shoulders, the apparatus was fixed to prevent humeral rotation (group 2). All repairs were cyclically loaded from 0 to 60 N at a displacement rate of 1 mm/s for 30 cycles. The constructs were then loaded to failure. Repair strength, gap formation, and strain were compared between groups. RESULTS: Cyclic loading revealed no difference in linear stiffness between testing conditions. Hysteresis was significantly greater when dynamic external rotation was allowed to occur. With load to failure, there were no differences in yield or ultimate load. Anterior tendon gap formation was greater at end rotation (30 degrees of humeral external rotation) and at yield load, and strain on the posterior tendon was less with dynamic external rotation. With dynamic external rotation, gap formation and tendon strain were significantly greater in the anterior region of the supraspinatus tendon compared with the posterior region. DISCUSSION: External rotation using postoperative physiologic loads affects gap formation and tendon strain between anterior and posterior supraspinatus tendon regions. Previous testing models without humeral rotation may underestimate gap formation and anterior tendon strain and overestimate posterior tendon strain. CLINICAL RELEVANCE: Understanding regional differences with respect to these variables, depending on quality of repair, may provide the surgeon a framework from which to prescribe guidelines for postoperative rehabilitation.

Direct phosphorylation and activation of a Nim1-related kinase Gin4 by Elm1 in budding yeast.

In budding yeast, Gin4, a Nim1-related kinase, plays an important role in proper organization of the septin ring at the mother-bud neck, a filamentous structure that is critical for diverse cellular processes including mitotic entry and cytokinesis. How Gin4 kinase activity is regulated is not known. Here we showed that a neck-associated Ser/Thr kinase Elm1, which is important for septin assembly, is critical for proper modification of Gin4 and its physiological substrate Shs1. In vitro studies with purified recombinant proteins demonstrated that Elm1 directly phosphorylates and activates Gin4, which in turn phosphorylates Shs1. Consistent with these observations, acute inhibition of Elm1 activity abolished mitotic Gin4 phosphorylation and Gin4-dependent Shs1 modification in vivo. In addition, a gin4 mutant lacking the Elm1-dependent phosphorylation sites exhibited an impaired localization to the bud-neck and, as a result, induced a significant growth defect with an elongated bud morphology. Thus, Elm1 regulates the septin assembly-dependent cellular events by directly phosphorylating and activating the Gin4-dependent pathway(s).

Coupling morphogenesis to mitotic entry.

In eukaryotes, cyclin B-bound cyclin-dependent protein kinase 1 promotes mitotic entry but is held in check, in part, by Wee1 protein kinase. Timely mitotic entry in budding yeast requires inactivation of Swe1 (Wee1 ortholog). Perturbations of the septin collar at the bud neck lead to Swe1 stabilization, delaying the G(2)/M transition. Swe1 is recruited to the neck and hyperphosphorylated before ubiquitin-mediated degradation. Hsl1 kinase (Nim1 ortholog), a negative regulator of Wee1, is required for efficient Swe1 localization at the neck but seems not to phosphorylate Swe1. Here, we show that two other kinases targeted sequentially to the neck, Cla4/PAK and Cdc5/Polo, are responsible for stepwise phosphorylation and down-regulation of Swe1. This mechanism links assembly of a cellular structure to passage into mitosis.