High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry.

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of non-photochemical quenching (NPQ) has proven to be an effective strategy to increase photosynthetic performance. However, the potential to breed for improved NPQ and a complete understanding of the genetic basis of NPQ relaxation is lacking due to limitations of oversampling and data collection from field-grown crop plants. Building on previous reports, we present a high-throughput assay for analysis of NPQ relaxation rates in Glycine max (soybean) using pulse amplitude modulated (PAM) chlorophyll fluorometry. Leaf disks are sampled from field-grown soybeans before transportation to a laboratory where NPQ relaxation is measured in a closed PAM-fluorometer. NPQ relaxation parameters are calculated by fitting a bi-exponential function to the measured NPQ values following a transition from high to low light. Using this method, it is possible to test hundreds of genotypes within a day. The procedure has the potential to screen mutant and diversity panels for variation in NPQ relaxation, and can therefore be applied to both fundamental and applied research questions.

Real-world effectiveness of anti-TNF switching in psoriatic arthritis: a systematic review of the literature.

Anti-tumor necrosis factors (Anti-TNFs) are a class of biologic disease-modifying anti-rheumatic drugs indicated for the treatment of moderate-to-severe psoriatic arthritis (PsA). Refractory patients are commonly managed by switching from one anti-TNF to another. To assess the evidence on the effectiveness of anti-TNF cycling in PsA patients, a systematic review of the literature was conducted. MEDLINE- and Embase-indexed English-language publications were systematically searched from 1995 to 2015 for studies assessing real-world effectiveness outcomes of anti-TNF cycling in PsA patients. Of 1086 citations identified, 18 studies were included; most conducted in Europe. Six of seven studies testing between lines found significant differences in effectiveness between earlier and subsequent lines of anti-TNF therapy. First-line therapy yielded better results compared with second-line therapy, and significant differences were observed between second- and third-line anti-TNF treatments. In the only study with multivariate regression testing for predictors of response, Danish registry patients were less likely to respond (American College of Rheumatology 20 % or 50 % response) to a second anti-TNF course if safety, rather than lack of effect, caused them to switch (odds ratio [OR] 0.04; p = 0.003 and OR 0.05; p = 0.03, respectively). Effectiveness of anti-TNFs at second line and later is reported in a small number of real-world studies of PsA patients. Subsequent treatment lines may be associated with less response in some measures. More research is needed to quantify the effectiveness of sequential anti-TNF lines in this progressive population' and to compare these effects with responses to drugs with different mechanisms of action.

Characteristics and temporal trends in patient registries: focus on the life sciences industry, 1981-2012.

PURPOSE: Patient registries are used to monitor safety, examine real-world effectiveness, and may potentially contribute to comparative effectiveness research. To our knowledge, life sciences industry (LSI)-sponsored registries have not been systematically categorized. This study represents a first step toward understanding such registries over time. METHODS: Studies described as registries were identified in the ClinicalTrials.gov database. Characteristics from these registry records were abstracted and analyzed. RESULTS: Of 1202 registries identified, approximately 47% reported LSI sponsorship. These 562 LSI registries varied in focus: medical devices (n = 193, 34%), specific drugs (n = 173, 31%), procedures (n = 29, 5%), or particular diseases (n = 139, 25%). Thirty-three registries (<6%) evaluated pregnancy outcomes. The most common therapeutic area was cardiovascular (n = 234, 42%); others included endocrinology, immunology, oncology, musculoskeletal disorders, and neurology. The two most often measured outcomes were clinical effectiveness and safety, each of which appeared in 363/562 (65%) of LSI registries. Other outcomes included real-world clinical practice patterns (n = 122, 22%), patient-reported outcomes (n = 106, 19%), disease epidemiology/natural history (n = 69, 12%), and economic outcomes (n = 30, 5%). The number of LSI registries and their geographic diversity has increased over time. CONCLUSIONS: The LSI registries represent a substantial proportion of all patient registries documented in ClinicalTrials.gov. These prospective studies are growing in number and encompass diverse therapeutic areas and geographic regions. Most registries measure multiple outcomes and capture real-world data that may be unavailable through other study designs. This classification of LSI registries documents their use for studying heterogeneity of diseases, examining treatment patterns, measuring patient-reported outcomes, examining economic outcomes, and performing comparative effectiveness research.

A Landscape-based model for predicting Mycobacterium ulcerans infection (Buruli Ulcer disease) presence in Benin, West Africa.

Mycobacterium ulcerans infection (Buruli ulcer [BU] disease) is an emerging tropical disease that causes severe morbidity in many communities, especially those in close proximity to aquatic environments. Research and control efforts are severely hampered by the paucity of data regarding the ecology of this disease; for example, the vectors and modes of transmission remain unknown. It is hypothesized that BU presence is associated with altered landscapes that perturb aquatic ecosystems; however, this has yet to be quantified over large spatial scales. We quantified relationships between land use/land cover (LULC) characteristics surrounding individual villages and BU presence in Benin, West Africa. We also examined the effects of other village-level characteristics which we hypothesized to affect BU presence, such as village distance to the nearest river. We found that as the percent urban land use in a 50-km buffer surrounding a village increased, the probability of BU presence decreased. Conversely, as the percent agricultural land use in a 20-km buffer surrounding a village increased, the probability of BU presence increased. Landscape-based models had predictive ability when predicting BU presence using validation data sets from Benin and Ghana, West Africa. Our analyses suggest that relatively small amounts of urbanization are associated with a decrease in the probability of BU presence, and we hypothesize that this is due to the increased availability of pumped water in urban environments. Our models provide an initial approach to predicting the probability of BU presence over large spatial scales in Benin and Ghana, using readily available land use data.

The location in cartilage of infectious retrovirus in cats infected with feline leukemia virus.

BACKGROUND: Previous studies have suggested that articular cartilage allografts were not likely to transmit infectious retrovirus since viral DNA could not be isolated from chondrocytes of infected individuals. However, the ability of the extracellular matrix of articular cartilage to harbor and transmit a retrovirus has not been examined. We hypothesized that articular cartilage fragments, but not isolated chondrocytes, from cats systemically infected with feline leukemia virus (FeLV) are capable of transmitting infectious retrovirus. METHODS: Fresh cartilage segments and chondrocytes isolated from cats systemically infected with feline leukemia virus were used in this study. Feline embryonic fibroblast cells were cocultured with segments of cartilage, isolated chondrocytes, or fragments of cortical bone from each infected cat. The FeLV p27 antigen was measured in the coculture media by enzyme-linked immunosorbent assay. In addition, FeLV proviral nucleic acids were quantified by real-time quantitative polymerase chain reaction with use of DNA extracted from feline embryonic fibroblast cell cocultures as well as isolated chondrocytes. Immunohistochemistry was used to assess for FeLV p27 antigen in both intact cartilage fragments and isolated chondrocytes. RESULTS: Feline embryonic fibroblast cells cocultured with cartilage fragments from each of the five FeLV-infected cats all demonstrated high levels of proviral DNA, indicating transmission of infective virus. In addition, media from all cocultures of feline embryonic fibroblast cells and chondral fragments became positive for p27 antigen, indicating active viral replication. In contrast, cocultures of feline embryonic fibroblast cells and isolated chondrocytes from all FeLV-infected cats were negative for proviral DNA and p27 antigen. Likewise, no proviral nucleic acids could be detected in isolated chondrocytes from any infected cats. Cocultures of feline embryonic fibroblast cells with cortical bone fragments were positive for proviral DNA and p27 antigen. Immunohistochemical staining of cartilage fragments from FeLV-infected cats demonstrated the presence of p27 antigen throughout the extracellular matrix, but the p27 antigen was not detected in isolated chondrocytes. CONCLUSIONS: Articular cartilage fragments can readily transmit infectious retrovirus, but isolated chondrocytes were likely not the source of the infectious virus because they did not harbor proviral DNA or p27 antigen.

Biomechanical evaluation of 2 techniques for ulnar collateral ligament reconstruction of the elbow.

BACKGROUND: Elbow medial ulnar collateral ligament tears often result in pain and instability that may be career threatening in overhead-throwing athletes. Surgical reconstruction is frequently chosen to treat this injury. Ulnar collateral ligament reconstruction as described by Jobe is the most commonly used technique. Testing of this construct has not demonstrated that the biomechanical parameters of the native ligament are restored. A more recent construct, the docking technique, may more reliably reproduce these factors. HYPOTHESIS: Increasing the number of strands of palmaris longus tendon graft used in ulnar collateral ligament reconstruction and tensioning them using the docking technique result in a construct with improved biomechanical parameters as compared with the Jobe technique. STUDY DESIGN: Controlled laboratory study. METHODS: Thirty-three fresh-frozen human cadaveric elbows were randomized into 3 subgroups: Jobe (11), docking (12), and native (10). The Jobe and docking groups underwent reconstruction using their described palmaris tendon graft constructs. The ulnar collateral ligament was left intact in the native group. Elbows were potted and tested using a servohydraulic materials testing machine to apply a valgus moment at 30 degrees of elbow flexion. Maximal moments to failure, stiffness, and strain at maximal moment and with a 3 N.m force applied were determined using a 2-camera motion analysis system to track reflective markers spanning the site. RESULTS: The docking (14.3 N.m) and native (18.8 N.m) subgroups resulted in higher maximal moment to failure than did the Jobe (8.9 N.m) subgroup (P < .001). There was no significant difference between native and docking groups (P > .05). Native ligaments were stiffer (301.4 N.m) than were Jobe (74.3 N.m) or docking (80.8 N.m; P < .001). Native ligaments demonstrated lower strain at maximal force (0.087 mm/mm) and 3 N.m forces (0.030 mm/mm) than did the Jobe (0.198/0.057 mm/mm) or docking (0.287/0.042 mm/mm) subgroups. There was no difference in stiffness or strain between the Jobe and docking subgroups (P > .05). CONCLUSION: Neither technique reproduced the biomechanical profile of the native ulnar collateral ligament; the findings of this study suggest that the docking construct may offer initial biomechanical advantage over the Jobe construct.

Isolated fibrillar damage in tendons stimulates local collagenase mRNA expression and protein synthesis.

The etiology of repetitive stress injuries in tendons has not been clearly identified. While minor trauma has been implicated as an inciting factor, the precise magnitude and structural level of tissue injury that initiates this degenerative cascade has not been determined. The purpose of this study was to determine if isolated tendon fibril damage could initiate an upregulation of interstitial collagenase (MMP13) mRNA and protein in tendon cells associated with the injured fibril(s). Rat tail tendon fascicles were subjected to in vitro tensile loading until isolated fibrillar damage was documented. Once fibrillar damage occurred, the tendons were immediately unloaded to 100g and maintained at that displacement for 24h under tissue culture conditions. In addition, non-injured tendon fascicles were maintained under unloaded (stress-deprived) conditions in culture for 24h to act as positive controls. In situ hybridization or immunohistochemistry was then performed to localize collagenase mRNA expression or protein synthesis, respectively. Fibrillar damage occurred at a similar stress (41.13+/-5.94MPa) and strain (13.24+/-1.94%) in the experimental tendons. In situ hybridization and immunohistochemistry demonstrated an upregulation of interstitial collagenase mRNA and protein, respectively, in only those cells associated with the damaged fibril(s). In the control (stress-deprived) specimens, collagenase mRNA expression and protein synthesis were observed throughout the fascicle. The results suggest that isolated fibrillar damage and the resultant upregulation of collagenase mRNA and protein in this damaged area occurs through a mechanobiological understimulation of tendon cells. This collagenase production may weaken the tendon and put more of the extracellular matrix at risk for further damage during subsequent loading.

Cartilage tolerates single impact loads of as much as half the joint fracture threshold.

We hypothesized that one mechanical insult could affect cellular proliferation, matrix turnover, and the structural integrity of cartilage, and that these effects would be dose dependent and time dependent. One impact load of low impact (14.4 MPa +/- 2.1 MPa), medium impact (22.8 MPa +/- 5.8 MPa), or high impact (55.5 MPa +/- 12.6 MPa) was administered to the posterior aspect of the medial femoral condyle of New Zealand White rabbits using a previously validated pendulum device. Animals were euthanized at 2, 6, and 12 weeks after impact, and the impacted and sham (contralateral limb) cartilage were harvested. Each specimen was assessed by light microscopy and by immunohistochemical methods. Although impacted specimens had greater loss of proteoglycan staining than sham cartilage, these changes were neither dose dependent nor time dependent. No structural damage, enzymatic proteoglycan or collagen breakdown, or cellular proliferation was identified in the different impact groups. Articular cartilage is a resilient tissue, particularly in situ, and can tolerate single impact loads of as much as 45% of the joint fracture threshold without considerable disruption or degradation.

Tendon injury response: assessment of biomechanical properties, tissue morphology and viability following flexor digitorum profundus tendon transection.

Insertion site injuries of the flexor digitorum profundus (FDP) tendon often present for delayed treatment. Apart from gross observations made at the time of surgery, the changes that occur in the flexor tendon stump during the interval from injury to repair are unknown. These changes may include tendon softening and loss of viability, which may contribute to the poor outcomes observed clinically and experimentally. Thirty-eight FDP tendons from 23 adult dogs were transected sharply from their insertions on the distal phalanges and were not repaired. Dogs were allowed full weight bearing and were euthanized 7 or 21 days after injury. Biomechanical testing indicated that the resistance of injured tendons to pullout of a Kessler-type suture was not different from control tendons at 7 days and was increased at 21 days by 25% (p<0.05). Morphologically, at 7 and 21 days the cut surface had a smooth appearance and the end of the injured tendon was increased in thickness by 30% and 50%, respectively (p<0.05). Histologically, we observed increased cellularity and dramatic fibroblast proliferation within the injured tendon stump; there was no evidence of decreased cell viability. We conclude that during the interval from 0 to 21 days after FDP insertion-site injury, tendons cells are viable, proliferative and synthesizing new matrix. This leads to increased tendon size and enhanced resistance to suture pullout. These findings offer a scientific rationale supporting the clinical practice of surgical re-attachment within the first 3 weeks after injury.

Induction of chondrocyte apoptosis following impact load.

OBJECTIVE: To investigate the presence and extent of chondrocyte apoptosis following impact load of articular cartilage in an in vivo model. DESIGN: An in vivo animal model, using a pendulum device and New Zealand White rabbits, was designed to study the effects of impact load on the development of chondrocyte apoptosis. Animals were placed into either a High Impact group or a Low Impact group, and the right medial femoral condyle was impacted with a single impact load. A sham operation was performed on the left limb, and this cartilage served as the control. SETTING: Academic medical center. PARTICIPANTS: New Zealand White rabbits (3 months). INTERVENTION: Impact load to the right medial femoral condyle. MAIN OUTCOME MEASURES: Three different methods were used to assess the presence and extent of chondrocyte apoptosis: 1) light microscopy (hematoxylin and eosin and terminal dUTP nick end labeling staining); 2) transmission electron microscopy; and 3) fluorescent microscopy with Hoechst 33342 staining. Secondary outcome measures included determination of the magnitude of impact force and time to peak force. RESULTS: Light microscopy demonstrated chondrocytes with changes consistent with apoptosis including condensed nuclei, deep eosinophilic cytoplasmic staining, and vacuolization within the impacted specimens. Terminal dUTP nick end labeling staining-stained specimens had a high degree of positively stained cells (60%) in both injured and uninjured specimens. Transmission electron microscopy of the impacted specimens demonstrated numerous chondrocytes with changes characteristic of apoptosis, including nuclear and cellular fragmentation, volume shrinkage, and cytoplasmic vacuolization. Eleven percent of the cells in the High Impact group had changes consistent with apoptosis, versus 3% for the low impact group and <1% for the sham specimens. The High Impact group received a statistically significant greater stress than the Low Impact group. Impact group (P < 0.05), and the average time to peak force was 0.021 seconds for each impact group. CONCLUSIONS: The current data strongly indicate that in vivo chondrocyte apoptosis can be stimulated by the application of a single, rapid impact load and that the extent of chondrocyte apoptosis is related to the amount of load applied. The contribution chondrocyte apoptosis makes to the development of posttraumatic arthritis following joint injury or intra-articular fracture still remains to be determined.

The insertion site of the canine flexor digitorum profundus tendon heals slowly following injury and suture repair.

Treatment of injuries of the flexor digitorum profundus (FDP) tendon insertion site has changed little during the past 50 years, in part because there are no reports describing flexor tendon insertion site healing. Our objective was to assess the effects of repair technique and post-operative time on tendon-bone healing using a canine model of injury and repair. We transected 48 FDP tendons from 24 dogs at their insertions and repaired them using either a four- or eight-strand suture technique. We assessed the mechanical properties of the repaired tendon-bone construct, tendon collagen biochemistry, and distal phalanx bone mineral density (BMD) at 0, 10, 21 and 42 days. Suture method had no significant effect on any outcome (p > 0.05). In particular, use of an eight-strand double modified Kessler technique did not result in increased stiffness or strength compared to a four-strand technique. With time, the repair site became stiffer, as demonstrated by a 230% increase in rigidity and a 50% decrease in strain from 0 to 42 days. However, from 0 to 42 days the ultimate force of the insertion site did not increase. This lack of increase in ultimate force was consistent with decreases in collagen content, non-reducible crosslinks and distal phalanx BMD. Taken together, our results indicate that the canine FDP tendon heals slowly after it is injured at its insertion site and sutured onto the distal phalanx. While these findings may be limited to the particular repair method we used, they demonstrate a need for devising new treatment strategies to improve healing of flexor tendon insertion site injuries.

The rigidity of repaired flexor tendons increases following ex vivo cyclic loading.

Transected flexor tendons are typically treated by suture repair followed by rehabilitation that generates repetitive tendon loading. Recent results in an in vivo canine model indicate that during the first 10 days after injury and repair, there is an increase in the rigidity of the tendon repair site. Our objective was to determine whether or not ex vivo cyclic loading of repaired flexor tendons causes a similar increase in repair-site rigidity. We simulated 10 days of rehabilitation by applying 6000 loading cycles to repaired canine flexor tendons ex vivo at force levels generated during passive motion rehabilitation; we then evaluated their tensile mechanical properties. High-force (peak force, 17 N) cyclic loading increased repair-site rigidity by 100% and decreased repair-site strain by 50%, whereas low-force (5 N) loading did not change the properties of the repair site. This mechanical conditioning effect may explain, in part, the changes in tensile properties observed after only 10 days of healing in vivo. Mechanical conditioning of repaired flexor tendons by repetitive forces applied during rehabilitation may lead to increases in repair-site rigidity and decreases in strain, thereby altering the mechanical loading environment of tissues and cells at the repair site.

A method for delivering variable impact stresses to the articular cartilage of rabbit knees.

OBJECTIVE: To develop a method by which a single impact force of controlled magnitude and rate could be applied uniformly to an area on the posterior aspect of the medial femoral condyle of adult rabbits. DESIGN: An in-vivo animal model using a pendulum device, designed and manufactured to supply the kinetic energy necessary to apply different impact loads to the posterior aspect of the medial femoral condyle of a rabbit. SETTING: Biomechanical laboratory, University Medical Center. SUBJECTS: A total of thirty-six femoral condyles from 3-kilogram New Zealand White (NZW) rabbits were used during this evaluation. INTERVENTION: An aluminum impactor was made based on the sagittal and coronal radii of curvature of six matched pairs (n = 12) of femurs from three-kilogram NZW rabbits. This impactor was coupled with the pendulum and used to apply different impact loads to both of the medial femoral condyle of the knees of NZW rabbits (n = 24). MAIN OUTCOME MEASUREMENTS: Peak impact force, time to peak impact force, and average contact area between impactor and medial femoral condyle, were measured for each group of animals tested. RESULTS: The pendulum delivered a consistent impact force to the rabbit condyle of 120.0 N (+/-18.1; coefficient of variance, 15 percent) with 400 grams attached to the pendulum arm, at an average time to peak force of 0.021 seconds (+/-0.001, co. var. 4.8 percent). The peak impact force was significantly different for each of the three impact mass groups of animals (p < 0.001). By contrast, time to peak force for each mass group averaged approximately 0.020 seconds and the average contact area was 6.26 mm2 (+/-0.51). Quantitative assessment of the exposed medium pressure-sensitive film confirmed uniform impact force intensity within each specimen. CONCLUSIONS: An in-vivo animal model was developed to deliver a controlled and rapid impact force to a specific area of the weight-bearing surface of the adult rabbit knee. These loads were applied at a rate comparable to the clinical setting of falling onto an outstretched hand, thus simulating a common clinical scenario by which cartilage is often injured. This model can be used in future experiments to investigate mechanism by which posttraumatic arthritis develops after articular injuries.